Methods and compositions for the delivery of bioactive compounds

ABSTRACT

The present invention provides compositions comprising delivery systems comprising a lipid vehicle, a bioactive compound, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the lipid vehicle encapsulates the polynucleotide of interest and the polyanionic carrier macromolecule, and wherein the delivery system is essentially free of carrier polynucleotides. Compositions also include delivery systems comprising a lipid vehicle and a bioactive agent, wherein the lipid vehicle encapsulates the bioactive agent, and wherein the lipid vehicle comprises a covalent bilayer or a core supported bilayer comprising polycationic lipids. Other compositions provided herein comprise a delivery system comprising a polypeptide of interest and a means for delivering the polypeptide into a cell. Also provided herein are methods for making the delivery systems, methods for delivering bioactive compounds, methods for treating a disease or unwanted condition in a subject with the delivery systems, and methods for detecting apoptosis in a cell.

FIELD OF THE INVENTION

The present invention relates to lipid vehicles for the delivery of bioactive compounds into cells.

BACKGROUND OF THE INVENTION

The development of new forms of therapeutics which use macromolecules such as proteins or nucleic acids as therapeutic agents has created a need to develop new and effective means of delivering such macromolecules to their appropriate cellular targets. Lipid-comprising vehicles have been developed to aid in the delivery of polynucleotides and other macromolecules. However, many of these vehicles exhibit undesirable pharmacokinetic properties and are associated with toxic side effects due to the stimulation of an inflammatory response when administered in vivo. Considering the great potential of novel macromolecular therapeutics in the treatment of various disorders, the need exists for the development of stable, bioavailable vehicles that are able to effectively deliver these therapeutics with minimal immunogenic effect.

BRIEF SUMMARY OF THE INVENTION

The presently disclosed subject matter provides compositions comprising delivery systems comprising a lipid vehicle, a bioactive compound, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the lipid vehicle encapsulates the bioactive compound and the polyanionic carrier macromolecule, and wherein the delivery system is essentially free of carrier polynucleotides. As most carrier polynucleotides (e.g., plasmid DNA, calf thymus DNA) can be immunogenic when delivered to a subject, the delivery systems disclosed herein that are essentially free of carrier polynucleotides have a reduced immunogenic effect when administered to a subject than those that comprise carrier polynucleotides.

Compositions also include delivery systems comprising a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, and wherein the lipid vehicle comprises a covalent bilayer or a core supported bilayer comprising polycationic lipids. In some of the embodiments wherein the lipid vehicle comprises a covalent bilayer, the covalent bilayer comprises a covalent leaflet supported bilayer. Covalent bilayers and supported bilayers provide stabilize the delivery system, allowing the surface charge of the lipid vehicle of the delivery system to be shielded fully or partially through the association of polyethylene glycol molecules with the surface of the lipid vehicle, thus reducing the clearance of the delivery system when administered to a subject and enhancing the bioavailability.

Other compositions provided herein comprise a delivery system comprising a polypeptide and a means for delivering the polypeptide into a cell, wherein the polypeptide can be used to detect apoptosis in a cell. In some of these embodiments, the polypeptide comprises at least one caspase 3 recognition sequence and a first amino acid residue conjugated to a donor fluorophore and a second amino acid residue conjugated to an acceptor fluorophore. Compositions further comprise an isolated polypeptide comprising at least one caspase 3 recognition sequence and a first amino acid residue conjugated to a donor fluorophore and a second amino acid residue conjugated to an acceptor fluorophore.

Also provided herein are methods of making the delivery systems, methods for delivering bioactive compounds, methods of treating a disease or unwanted condition with the delivery systems comprising a bioactive compound that has therapeutic activity against the disease or unwanted condition, and methods of detecting apoptosis in a cell.

As such, in some embodiments the presently disclosed subject matter provides delivery systems comprising a cationic liposome, a bioactive compound, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein said cationic liposome encapsulates said bioactive compound and said polyanionic carrier macromolecule, wherein said cationic liposome comprises a lipid bilayer that comprises an inner leaflet and an outer leaflet, and wherein said delivery system is essentially free of carrier polynucleotides. In some embodiments, said polyanionic carrier macromolecule comprises a polyanionic carrier polysaccharide, a polyanionic carrier polypeptide, or a combination thereof. In some embodiments, said polyanionic carrier polysaccharide comprises a glycosaminoglycan. In some embodiments, said glycosaminoglycan is selected from the group consisting of heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, and dextran sulfate. In some embodiments, said glycosaminoglycan comprises hyaluronic acid. In some embodiments, said polyanionic carrier polypeptide comprises a poly-glutamic acid or a poly-aspartic acid.

In some embodiments, the presently disclosed subject matter also provides delivery systems comprising a lipid vehicle and a bioactive compound, wherein said lipid vehicle encapsulates said bioactive compound, wherein said lipid vehicle comprises a core supported bilayer, wherein said core supported bilayer comprises an inner leaflet and an outer leaflet, and wherein said core supported bilayer comprises a polycationic lipid. In some embodiments, said lipid vehicle comprises a cationic liposome. In some embodiments, the presently disclosed delivery systems further comprise a polyanionic carrier macromolecule, wherein said lipid vehicle encapsulates said polyanionic carrier macromolecule.

The presently disclosed subject matter also provides delivery systems comprising a lipid vehicle and a bioactive compound, wherein said lipid vehicle encapsulates said bioactive compound, and wherein said lipid vehicle comprises a covalent bilayer, wherein said covalent bilayer comprises an inner leaflet and an outer leaflet. In some embodiments, said covalent bilayer comprises an inner leaflet comprising an amphipathic polymer, wherein said amphipathic polymer comprises a polymeric backbone, wherein said polymeric backbone comprises a chain of repeating monomeric units bound to one another by a covalent bond, wherein at least 10% of said monomeric units within said polymeric backbone comprise a side chain and wherein said amphipathic polymer is selected from the group consisting of (a) an amphipathic polymer, wherein said monomeric units of said polymeric backbone comprise hydrophilic monomeric units, and wherein said side chains of said monomeric units comprise hydrophobic side chains; (b) an amphipathic polymer, wherein said monomeric units of said polymeric backbone comprise hydrophobic monomeric units, and wherein said side chains of said monomeric units comprise hydrophilic side chains; and (c) an amphipathic polymer, wherein said monomeric units of said polymeric backbone comprise hydrophobic or hydrophilic monomeric units, and wherein said side chains alternate between hydrophobic and hydrophilic side chains, thereby forming an alternating amphipathic polymer. In some embodiments, said inner leaflet comprises an alternating amphipathic polymer.

In some embodiments of the presently disclosed delivery systems, said covalent bilayer of said lipid vehicle comprises a biocleavable covalent bond.

In some embodiments of the presently disclosed delivery systems, said outer leaflet comprises a plurality of cationic lipids, wherein said outer leaflet does not comprise covalent bonds between said cationic lipids.

In some embodiments, the presently disclosed delivery systems further comprise a polycation, wherein said cationic liposome encapsulates said polycation. In some embodiments, said polycation comprises a polycationic polypeptide. In some embodiments, said polycationic polypeptide comprises protamine.

In some embodiments of the presently disclosed delivery systems, said outer leaflet comprises a lipid-polyethylene glycol (lipid-PEG) conjugate. In some embodiments, said delivery system comprises a stealth delivery system. In some embodiments, said outer leaflet comprises a lipid-PEG conjugate at a concentration of about 8 mol % to about 12 mol % of the total lipids. In some embodiments, said concentration is about 10.6 mol % of the total lipids. In some embodiments, said lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In some embodiments, said lipid-PEG conjugate comprises a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol2000 (DSPE-PEG2000).

In some embodiments of the presently disclosed delivery systems, said outer leaflet comprises a targeting ligand, thereby forming a targeted delivery system, wherein said targeting ligand targets said targeted delivery system to a targeted cell. In some embodiments, said targeting ligand comprises a benzamide derivative. In some embodiments, said benzamide derivative comprises anisamide. In some embodiments, said anisamide is conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG), thereby producing DSPE-PEG-AA. In some embodiments, said targeted cell comprises a cancer cell. In some embodiments, said cancer is selected from the group consisting of a bladder cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, an endometrial cancer, a hepatocellular carcinoma, a laryngeal cancer, a lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, and a thyroid cancer. In some embodiments, said cancer comprises a lung cancer.

In some embodiments of the presently disclosed delivery systems, said bioactive compound comprises a polynucleotide of interest. In some embodiments, said polynucleotide of interest comprises a silencing element, wherein expression or introduction of said silencing element into a cell reduces the expression of a target polynucleotide or the polypeptide encoded thereby. In some embodiments, said silencing element comprises an interfering RNA. In some embodiments, said interfering RNA comprises an siRNA. In some embodiments, said target polynucleotide comprises an oncogene. In some embodiments, said oncogene comprises an epidermal growth factor receptor.

In some embodiments of the presently disclosed delivery systems, said bioactive compound comprises a polypeptide of interest. In some embodiments, said polypeptide of interest comprises an anionic polypeptide. In some embodiments, said anionic polypeptide has the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, said polypeptide of interest has at least one caspase 3 recognition motif and a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest, wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source. In some embodiments, said polypeptide of interest has the sequence set forth in SEQ ID NO: 6. In some embodiments, said polypeptide of interest is acetylated at its amino terminus. In some embodiments, said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or wherein said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue. In some embodiments, said donor fluorophore comprises Cy5.5 and said acceptor fluorophore comprises Cy7. In some embodiments, the presently disclosed delivery systems further comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.

The presently disclosed subject matter also provides delivery systems comprising a polypeptide of interest and a means for delivering said polypeptide of interest into a cell, wherein said polypeptide of interest has at least one caspase 3 recognition motif and a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest, wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source. In some embodiments, said polypeptide of interest comprises an anionic polypeptide. In some embodiments, said polypeptide of interest has the sequence set forth in SEQ ID NO: 6. In some embodiments, said polypeptide of interest is acetylated at its amino terminus. In some embodiments, said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or wherein said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue. In some embodiments, said donor fluorophore comprises Cy5.5 and said acceptor fluorophore comprises Cy7. In some embodiments, said means for delivering said polypeptide of interest comprises a lipid vehicle, wherein said lipid vehicle encapsulates said polypeptide of interest.

In some embodiments, the presently disclosed delivery systems further comprise a cytotoxic bioactive compound, wherein said lipid vehicle encapsulates said cytotoxic bioactive compound. In some embodiments, said cytotoxic bioactive compound comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, said lipid vehicle comprises a cationic liposome.

In some embodiments, the presently disclosed delivery systems further comprise a polyanionic carrier macromolecule, wherein said lipid vehicle encapsulates said polyanionic carrier macromolecule. In some embodiments, said polyanionic carrier macromolecule comprises a polyanionic carrier polysaccharide, a polyanionic carrier polypeptide, or a combination thereof. In some embodiments, said polyanionic carrier polysaccharide comprises a glycosaminoglycan. In some embodiments, said glycosaminoglycan is selected from the group consisting of heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, and dextran sulfate. In some embodiments, said glycosaminoglycan comprises heparin sulfate.

In some embodiments, the presently disclosed delivery systems further comprise a polycation, wherein said lipid vehicle encapsulates said polycation. In some embodiments, said polycation comprises a polycationic polypeptide. In some embodiments, said polycationic polypeptide comprises protamine.

In some embodiments, of the presently disclosed delivery systems, said lipid vehicle comprises an exterior surface, and wherein said exterior surface comprises a polyethylene glycol (PEG) molecule. In some embodiments, said delivery system comprises a stealth delivery system. In some embodiments, said PEG molecule comprises a lipid-PEG conjugate, wherein said lipid vehicle comprises a liposome, wherein said liposome comprises an inner leaflet and an outer leaflet, and wherein said outer leaflet comprises a lipid-PEG conjugate at a concentration of about 8 mol % to about 12 mol % of the total lipids. In some embodiments, said concentration is about 10.6 mol % of the total lipids. In some embodiments, said lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In some embodiments, said lipid-PEG conjugate comprises a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol2000 (DSPE-PEG2000).

In some embodiments of the presently disclosed delivery systems, said lipid vehicle of said delivery system comprises an exterior surface, wherein said exterior surface comprises a targeting ligand, thereby forming a targeted delivery system, wherein said targeting ligand targets said targeted delivery system to a targeted cell. In some embodiments, said targeting ligand comprises a benzamide derivative. In some embodiments, said benzamide derivative comprises anisamide. In some embodiments, anisamide is conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG), thereby producing DSPE-PEG-AA. In some embodiments, said targeted cell comprises a cancer cell. In some embodiments, said cancer is selected from the group consisting of a bladder cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, an endometrial cancer, a hepatocellular carcinoma, a laryngeal cancer, a lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, and a thyroid cancer. In some embodiments, said cancer comprises a lung cancer.

The presently disclosed subject matter also provides in some embodiments pharmaceutical compositions comprising the presently disclosed delivery systems and a pharmaceutically acceptable carrier.

In some embodiments, the presently disclosed subject matter also provides isolated polypeptides having the sequence set forth in SEQ ID NO: 6, wherein a donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein an acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or wherein an acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein a donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue. In some embodiments, said donor fluorophore comprises Cy5.5 and said acceptor fluorophore comprises Cy7.

The presently disclosed subject matter also provides in some embodiments methods for delivering bioactive compounds to cells. In some embodiments, said methods comprise contacting the cell with the presently disclosed delivery systems.

The presently disclosed subject matter also provides in some embodiments methods for treating diseases or unwanted conditions in subjects. In some embodiments, said methods comprise administering the presently disclosed pharmaceutical compositions to said subjects, wherein said bioactive compounds have therapeutic activities against said diseases and/or unwanted conditions. In some embodiments, said disease comprises a cancer.

The presently disclosed subject matter also provides in some embodiments methods for making delivery systems comprising a cationic liposome, a polynucleotide of interest, and a polyanionic carrier macromolecule, wherein said polyanionic carrier macromolecule does not comprise a carrier polynucleotide, and wherein said cationic liposome encapsulates said polynucleotide of interest and said polyanionic macromolecule. In some embodiments, the presently disclosed methods comprise (a) providing a cationic liposome; and (b) mixing said cationic liposome with a solution comprising a polynucleotide of interest and a polyanionic carrier macromolecule, thereby forming said delivery system. In some embodiments, said delivery systems further comprise a polycation, wherein said cationic liposome encapsulates said polycation, and wherein said solution further comprises a polycation. In some embodiments, step (b) produces a cationic liposome comprising a core supported bilayer.

In some embodiments, the presently disclosed subject matter also provides methods for making delivery systems comprising a cationic liposome, a polynucleotide of interest, a polyanionic carrier macromolecule, and a polycation, wherein said polyanionic carrier macromolecule does not comprise a carrier polynucleotide, and wherein said cationic liposome encapsulates said polynucleotide of interest, said polyanionic carrier macromolecule, and said polycation. In some embodiments, said methods comprise (a) providing a cationic liposome; (b) mixing said cationic liposome with a polycation to form a liposome/polycation solution; (c) mixing a polynucleotide of interest with a polyanionic carrier macromolecule to form a polynucleotide/polyanionic carrier macromolecule solution; and (d) mixing said polynucleotide/polyanionic carrier macromolecule solution with said liposome/polycation solution, thereby forming said delivery system; wherein steps (a) and (b) can be performed before or after step (c). In some embodiments, step (d) produces a cationic liposome comprising a core supported bilayer.

In some embodiments, the presently disclosed methods further comprise a post-insertion step, wherein at least one of a lipid-targeting ligand conjugate or a lipid-polyethylene glycol (lipid-PEG) conjugate is post-inserted into said cationic liposome, wherein said post-insertion step is performed following step a) or after forming said delivery system. In some embodiments, said delivery system comprises a stealth delivery system. In some embodiments, post-insertion step comprises incubating said delivery system with a lipid-PEG conjugate at a concentration of about 5 to about 20 mol %. In some embodiments, said concentration of said lipid-PEG conjugate comprises about 10 mol %. In some embodiments, said lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In some embodiments, said lipid-PEG conjugate comprises a 1,2-distearoyl-sn-glycero-5-phosphoethanolamine-N-carboxy-polyethylene glycol2000.

In some embodiments, the presently disclosed subject matter also provides methods for making delivery systems comprising a lipid vehicle and a bioactive compound, wherein said lipid vehicle encapsulates said bioactive compound, wherein said lipid vehicle comprises a core supported bilayer, and wherein said core supported bilayer comprises a polycationic lipid. In some embodiments, said methods comprise (a) providing a lipid vehicle comprising a polycationic lipid; and (b) mixing said lipid vehicle with a bioactive compound, thereby forming said delivery system, wherein said lipid vehicle of said delivery system comprises a core supported bilayer.

In some embodiments, the presently disclosed subject matter also provides methods for making a delivery system comprising a lipid vehicle and a bioactive compound, wherein said lipid vehicle encapsulates said bioactive compound, and wherein said lipid vehicle comprises a covalent bilayer. In some embodiments, said methods comprise (a) providing an amphipathic polymer in a solution comprising water, an immiscible, volatile organic solvent, and a bioactive compound, thereby forming a water-in-oil emulsion, wherein said amphipathic polymer comprises a polymeric backbone, wherein said polymeric backbone comprises a chain of repeating monomelic units bound to one another by a covalent bond, wherein at least 10% of said monomelic units within said polymeric backbone comprise a side chain, and wherein said amphipathic polymer is selected from the group consisting of (i) an amphipathic polymer, wherein said monomelic units of said polymeric backbone comprise hydrophilic monomeric units, and wherein said side chains of said monomeric units comprise hydrophobic side chains; (ii) an amphipathic polymer, wherein said monomeric units of said polymeric backbone comprise hydrophobic monomeric units, and wherein said side chains of said monomeric units comprise hydrophilic side chains; and (iii) an amphipathic polymer, wherein said monomeric units of said polymeric backbone comprise hydrophobic or hydrophilic monomeric units, and wherein said side chains alternate between hydrophobic and hydrophilic side chains, thereby forming an alternating amphipathic polymer; (b) mixing amphipathic lipids with said water-in-oil emulsion; and (c) evaporating said volatile organic solvent, thereby forming said delivery system.

In some embodiments, the presently disclosed subject matter also provides methods for making a delivery system comprising a lipid vehicle and a bioactive compound, wherein said lipid vehicle encapsulates said bioactive compound, and wherein said lipid vehicle comprises a covalent bilayer. In some embodiments, said methods comprise (a) mixing a plurality of amphipathic lipids, wherein the amphipathic lipids comprise a cross-linkable hydrophilic head group, in a solution comprising water, an immiscible, volatile organic solvent, and a bioactive compound, thereby forming a water-in-oil emulsion; (b) adding an initiator, thereby cross-linking the cross-linkable head groups of said amphipathic lipids; (c) mixing amphipathic lipids with said water-in-oil emulsion; and (d) evaporating said volatile organic solvent, thereby forming said delivery system. In some embodiments, said covalent bilayer comprises a covalent leaflet supported bilayer. In some embodiments, the presently disclosed methods further comprise a post-insertion step, wherein at least one of a lipid-targeting ligand conjugate and a lipid-polyethylene glycol (lipid-PEG) conjugate is post-inserted into said covalent bilayer, wherein said post-insertion step is performed after forming said delivery system. In some embodiments, said lipid-PEG conjugate comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG). In some embodiments, said lipid-targeting ligand conjugate comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol-anisamide (DSPE-PEG-anisamide).

The presently disclosed subject matter also provides in some embodiments methods for detecting apoptosis in cells. In some embodiments, said methods comprise (a) contacting said cell with a delivery system comprising a polypeptide of interest, wherein said polypeptide of interest has at least one caspase 3 recognition motif and a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest, wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source; (b) exciting said donor fluorophore with a light source; and (c) detecting the emission of said donor fluorophore and said acceptor fluorophore, wherein an increase in the ratio of the emission of said donor fluorophore to said acceptor fluorophore in comparison to a control cell indicates apoptosis in said cell. In some embodiments, said polypeptide of interest has the sequence set forth in SEQ ID NO: 6. In some embodiments, said polypeptide of interest is acetylated at its amino terminus. In some embodiments, said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue. In some embodiments, said donor fluorophore comprises Cy5.5 and said acceptor fluorophore comprises Cy7. In some embodiments, said means for delivering said polypeptide of interest into said cell comprises a lipid vehicle, wherein said lipid vehicle encapsulates said polypeptide of interest. In some embodiments, said delivery system further comprises a cytotoxic bioactive compound, wherein said lipid vehicle encapsulates said cytotoxic bioactive compound. In some embodiments, said cytotoxic bioactive compound comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, said lipid vehicle comprises a cationic liposome.

In some embodiments, the presently disclosed lipid vehicles further comprise a polyanionic carrier macromolecule, wherein said lipid vehicle encapsulates said polyanionic carrier macromolecule. In some embodiments, said polyanionic carrier macromolecule comprises a polyanionic carrier polysaccharide, a polyanionic carrier polypeptide, or a combination thereof. In some embodiments, said polyanionic carrier polysaccharide comprises a glycosaminoglycan. In some embodiments, said glycosaminoglycan is selected from the group consisting of heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, and dextran sulfate. In some embodiments, said glycosaminoglycan comprises heparin sulfate.

In some embodiments, the presently disclosed lipid vehicles further comprise a polycation, wherein said lipid vehicle encapsulates said polycation. In some embodiments, said polycation comprises a polycationic polypeptide. In some embodiments, said polycationic polypeptide comprises protamine.

In some embodiments, said lipid vehicle comprises an exterior surface, and wherein said exterior surface comprises a polyethylene glycol (PEG) molecule. In some embodiments, said delivery system comprises a stealth delivery system. In some embodiments, said PEG molecule comprises a lipid-PEG conjugate, wherein said lipid vehicle comprises a liposome, wherein said liposome comprises an inner leaflet and an outer leaflet, and wherein said outer leaflet comprises a lipid-PEG conjugate at a concentration of about 8 mol % to about 12 mol % of the total lipids. In some embodiments, said concentration is about 10.6 mol % of the total lipids. In some embodiments, said lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In some embodiments, said lipid-PEG conjugate comprises a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol2000 (DSPE-PEG2000).

In some embodiments of the presently disclosed subject matter, said lipid vehicles of said delivery systems comprise an exterior surface, wherein said exterior surface comprises a targeting ligand, thereby forming a targeted delivery system, wherein said targeting ligand targets said targeted delivery system to a targeted cell. In some embodiments, said targeting ligand comprises a benzamide derivative. In some embodiments, said benzamide derivative comprises anisamide. In some embodiments, said anisamide is conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG), thereby producing DSPE-PEG-AA.

In some embodiments of the presently disclosed methods, said targeted cell comprises a cancer cell. In some embodiments, said cancer is selected from the group consisting of a bladder cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, an endometrial cancer, a hepatocellular carcinoma, a laryngeal cancer, a lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, and a thyroid cancer. In some embodiments, said cancer comprises a lung cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Formula (II), which comprises the molecular structure of amphipol compounds that were described in Tribet, Audebert, and Popot (1996) Proc Natl Acad Sci USA 93:15047-15050. The weight average apparent molecular weight (<MW>) was deduced from the <MW> of the polyacrylate precursors as estimated by gel permeation chromatography using narrow MW polyoxyethylene calibration standards (which may entail systematic errors). DP (<MW>/MW of monomer) is the corresponding number of units per chain. The number of monomers per chain in the most abundant molecules (representing approximately 80% of the mass) actually ranges over one decade. x, y, and z are the molar percentages of each type of unit, randomly distributed along the chain. The average number of each type of unit per amphipol molecule is given between parentheses.

FIG. 2 shows the effect of (siRNA+hyaluronic acid (HA))/protamine weight ratio on particle size and zeta potential of siRNA/HA/protamine complexes. Protamine (200 μg/mL, 150 μl) and a mixture of siRNA and HA (160-210 μg/ml, weight ratio=1:1, 150 μl) were mixed in a 1.5 ml tube and kept at room temperature for ten minutes and the size and associated zeta potential of the resulting particle were measured.

FIG. 3 demonstrates the effect of lipid/siRNA molar ratio on particle size and zeta potential of lipid/polycation/HA nanoparticles (LPH-NPs). Complexes of siRNA/HA and protamine ((siRNA+HA)/protamine weight ratio=1.0, 300 μl) were mixed with 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP)/cholesterol liposomes (total lipid concentration=40 mM, 0-50 μl) and incubated at room temperature for ten minutes. The size and associated zeta potential of the resulting particle were measured.

FIG. 4 depicts the effect of the lipid/siRNA molar ratio of LPH-NP on in vitro intracellular siRNA delivery in B16F10 cells. LPH-NP containing 500 nM FAM-siRNA was added to B16F10 cells (0.5×10⁵ cells/well) and incubated for four hours, after which the fluorescence intensity was measured. Each value represents the mean±S.D. (n=4).

FIG. 5 presents a TEM image of LPH-NP with a lipid/siRNA molar ratio of 1067. Bar indicates 100 nm.

FIG. 6 shows the in vitro intracellular siRNA delivery of different formulations in B16F10 cells. Formulations containing FAM-siRNA (500 nM) were added to B16F10 cells (0.5×10⁵ cells/well) and then incubated at 37° C. for four hours, after which, fluorescence intensity was measured. For free ligand competition studies, cells were co-incubated with 50 μM haloperidol and the various formulations. LPD-NP:lipid/polycation/DNA nanoparticles. Each value represents the mean±S.D. (n=4). ^(a)) p<0.05, significantly different compared with the free siRNA. ^(b)) p<0.05, significantly different compared with the non-targeted LPH-NP. ^(c)) p<0.05, significantly different compared with the targeted LPH-NP. ^(d)) p<0.05, significantly different compared with the non-targeted LPD-NP.

FIG. 7 demonstrates the in vitro luciferase gene silencing effects of different formulations in B16F10 cells. Each formulation containing anti-luciferase siRNA (250 nM) was added to B16F10 cells (1×10⁵ cells/well) and then incubated at 37° C. for 24 hours, after which the luciferase activity was measured. Each value represents the mean±S.D. (n=3). ^(a)) p<0.05, significantly different compared with the untreated control. ^(b)) p<0.05, significantly different compared with the non-targeted LPH-NP. ^(c)) p<0.05, significantly different compared with the non-targeted LPD-NP.

FIGS. 8A and 8B present the in vivo luciferase gene silencing effects of different formulations at the dose of 0.15 mg/kg (FIG. 7A) and that of the targeted LPH-NP at various doses (FIG. 7B) in the pulmonary metastatic tumors. C57BL/6 mice were i.v. injected with 2×10⁵ B16F10 cells via the tail vein. Seventeen days later, mice were given i.v. injections of different siRNA formulations. After 24 hours, luciferase activity in lung tumors was measured. In FIG. 7B, closed circles represent luciferase siRNA in targeted LPH-NP and open circles represent control siRNA in targeted LPH-NP. Each value represents the mean±S.D. (n=3-4). *p<0.05, significantly different compared with the untreated control.

FIG. 9 provides the serum cytokine levels of C57BL/6 mice 2 h after i.v. injection of siRNA in various formulations. Blood was collected from the tail artery and serum IL6 (closed symbols) and IL12 (open symbols) levels were analyzed. Circles represent siRNA and HA in targeted LPH-NP, triangles represent siRNA and calf thymus DNA in targeted LPD-NP, squares represent siRNA and plasmid DNA in targeted LPD-NP. Each value represents the mean±S.D. (n=4). *p<0.05, significantly different compared with the untreated control (siRNA dose=0 mg/kg).

FIG. 10A provides a cryo-TEM photograph of LPD nanoparticles and was reproduced from Tan et al. (2002) Methods Mol Med 69:73-81 with the authors' permission. FIGS. 10B and 10C depict the structure of the double lipid bilayer and a proposed mechanism for the formation of LPD nanoparticles, respectively.

FIG. 11 depicts the chemical structures of the lipids N-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride (DOTAP) and N,N-di-stearoyl-N-methyl-N-2[N′—(N6-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DSGLA).

FIG. 12 provides the size distribution of different PEGylated formulations. Data=mean±SD, n=4-6.

FIG. 13 presents TEM photographs of liposomes/PEGylated liposomes and LPD/PEGylated LPD. Upper panel scale bar=200 nm, lower panel scale bar=100 nm. Arrows indicate the “sprouts” of the particles and arrow heads indicate the small particles.

FIGS. 14A-14D provide data from size exclusion chromatography of different samples. FIG. 14A describes the chromatography of particles of different sizes; FIG. 14B provides the chromatography of DOTAP in different formulations; FIG. 14C presents the chromatography of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG) in different formulations; and FIG. 14D presents the chromatography of different components in the PEGylated LPD. DOTAP liposomes were labeled with NBD-DOTAP; DSPE-PEG micelles were labeled with DSPE-PEG-CF; and siRNA was labeled with FAM-siRNA. Data are representative chromatography from 2-3 batches of formulations.

FIG. 15 provides the zeta potential of different formulations: A represents LPD; B represents LPD+10 mol % DSPE-PEG; and C represents LPD+10 mol % DSPE-PEG purified by the size exclusion column (first peak). Data=mean±SD, n=3

FIG. 16 shows the liver sinusoidal uptake of cy3-siRNA (red) in different LPD formulations. F-actin outlining the cellular morphology was stained with Alexa Fluor® 488 Phalloidin (green) and nuclei were stained with DAPI (blue). Magnification=1,600 x. Data are representative pictures from 3 mice in each group.

FIG. 17 depicts the proposed mechanism for the formation of PEGylated LPD nanoparticles with a core supported bilayer.

FIG. 18 illustrates a non-limiting method for preparing a delivery system with a covalent bilayer comprising an amphipathic polymer.

FIG. 19 depicts a non-limiting method for the preparation of a delivery system with a covalent bilayer comprising amphipathic lipids with cross-linked hydrophilic head groups.

FIGS. 20A and 20B show the inhibition of STAT5b phosphorylation by the EV peptide (SEQ ID NO: 3) in a tumor cell lysate. Increasing amounts of EV peptide was added to the assay mixture containing H460 cell lysate. The degree of STAT5b phosphorylation was detected by Western blot (FIG. 20A) using a phosphorylated STAT5b specific monoclonal antibody. The data was analyzed by Image J software to obtain the IC₅₀ (FIG. 20B).

FIG. 21 shows a graph depicting the uptake of the EV peptide by H460 cells. After treating H460 cells for four hours with an Alexa488 conjugated EV peptide formulated in LPH, PEG-LPH, or PEG-AA-LPH, cells were washed, treated with Triton X-100, and the fluorescence of the cell extract was determined with a fluorometer.

FIG. 22 provides a graph depicting the inhibition of STAT5b phosphorylation by the EV peptide. At 24 hours after treating H460 cells with LPH, LPH-PEG or LPH-PEG-AA comprising the EV (SEQ ID NO: 3) or EE (SEQ ID NO: 4) peptide or the free peptide at 3.4 μM, the reduction of phosphorylated Stat5b was assayed with an ELISA.

FIG. 23 shows a graph displaying the inhibition of cell growth by the EV peptide. H460 cells were treated with the EV peptide (5 μM) formulated in LPH-PEG-AA, LPH-PEG, or as a free peptide.

FIG. 24 depicts the changes in cell morphology that result from a 48 hour treatment of H460 cells with the EE or EV peptide formulated in LPH-PEG-AA. The upper panels are phase contrast images and the lower panels are fluorescence images after the culture was stained by DAPI for nucleus morphology observation.

FIGS. 25A-25E shows results from flow cytometry assays depicting peptide-induced apoptosis of H460 cells. Cells were stained with propidium iodide (PI) and Annexin V-FITC at 48 hours after treatment with a PEGylated LPH with AA or without AA. FIG. 25A shows untreated H460 cells. FIG. 25B shows cells treated with the EE peptide formulated in LPH-PEG. FIG. 25C shows cells treated with the EE peptide in LPH-PEG-AA. FIG. 25D shows cells treated with the EV peptide in LPH-PEG. FIG. 25E shows cells treated with the EV peptide in LPH-PEG-AA.

FIG. 26 depicts the uptake of the EV peptide by various organs and tumor. Alexa488-conjugated EV peptide (4 μg) was formulated in LPH, LPH-PEG, or LPH-PEG-AA nanoparticles and injected intravenously into H460 tumor-bearing mice. After 4 hours, major organs were excised and photographed with an IVIS optical camera.

FIG. 27A-27D show the emission spectra of the imaging peptide. The fluorescence emission profiles of untreated peptide (FIG. 27A), peptide treated with 60 units of caspase-3 for 2 hours (FIG. 27B), peptide treated with 0.1 mg BSA (FIG. 27C), and peptide treated with 60 units caspase-3 that was pre-incubated with 10 μM caspase-3 inhibitor for one hour (FIG. 27D) were measured with a nanofluorometer.

FIG. 28 depicts the fluorescence of H460 cells treated with the imaging peptide formulated with LPD-PEG-AA for 30 minutes. Cells were treated with Taxol (50 μM) to induce apoptosis. Cy5.5 and Cy7 were both excited by HeNe 633 nm laser. The Cy5.5 channel was 680-740 nm. The Cy7 channel was 740-850 nm. Boxes indicate the cells randomly selected for analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

Throughout this specification and the claims, the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant to encompass variations of, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

I. Compositions

Provided herein are delivery systems comprising a cationic liposome encapsulating a bioactive compound (e.g., polynucleotide of interest) and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the delivery system is essentially free of carrier polynucleotides.

The invention further provides delivery systems comprising a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, and wherein the lipid vehicle comprises a covalent bilayer or a core supported bilayer comprising a polycationic lipid. Other compositions include a delivery system comprising a polypeptide and a means for delivering the polypeptide into a cell, wherein the polypeptide comprises at least one caspase 3 recognition motif and a first amino acid conjugated to a donor fluorophore and a second amino acid conjugated to an acceptor fluorophore. Also provided herein are pharmaceutical compositions comprising the delivery systems and a pharmaceutically acceptable carrier.

A. Lipid Vehicles

As used herein, a “delivery system” or “delivery system complex” comprises a bioactive compound and a means for delivering the bioactive compound to a cell. In some embodiments, the delivery system comprises a lipid vehicle that encapsulates the bioactive compound. As used herein, “lipid vehicle” refers to a lipid composition that is capable of delivering a bioactive compound to a physiological site or cell. Lipid vehicles can include, but are not limited to, micelles, microemulsions, macroemulsions, and liposomes.

The term “micelles” refers to colloidal aggregates of amphipathic molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the nonpolar portions of the lipid molecules at the interior of the micelle and the polar portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) has a range from about 50 to about 100. The term “micelles” also refers to inverse or reverse micelles, which are formed in a nonpolar solvent, wherein the nonpolar portions are at the exterior surface, exposed to the nonpolar solvent and the polar portion is oriented towards the interior of the micelle.

As described herein, microemulsions are essentially swollen micelles, although not all micellar solutions can be swollen to form a microemulsion. Microemulsions are thermodynamically stable, are formed spontaneously, and contain particles that are extremely small. Droplet diameters in microemulsions typically range from about 10 to about 100 nm. In contrast, the term macroemulsions refers to droplets having diameters greater than about 100 nm.

Liposomes are self-assembling, substantially spherical vesicles comprising a lipid bilayer that encircles an aqueous interior or core, wherein the lipid bilayer comprises amphipathic lipids having hydrophilic headgroups and hydrophobic tails, in which the hydrophilic headgroups of the amphipathic lipid molecules are oriented toward the aqueous solution, while the hydrophobic tails orient toward the interior of the bilayer. The lipid bilayer structure thereby comprises two opposing monolayers that are referred to as the “inner leaflet” and the “outer leaflet,” wherein the hydrophobic tails are shielded from contact with the surrounding medium. The “inner leaflet” is the monolayer wherein the hydrophilic head groups are oriented toward the aqueous core of the liposome. The “outer leaflet” is the monolayer comprising amphipathic lipids, wherein the hydrophilic head groups are oriented towards the outer surface of the liposome. Liposomes typically have a diameter ranging from about 25 nm to about 1 μm. (see, e.g., Shah (ed.) (1998) Micelles, Microemulsions, and Monolayers: Science and Technology, Marcel Dekker; Janoff (ed.) (1998) Liposomes: Rational Design, Marcel Dekker). The term “liposome” encompasses both multilamellar liposomes comprised of anywhere from two to hundreds of concentric lipid bilayers alternating with layers of an aqueous phase and unilamellar vesicles that are comprised of a single lipid bilayer. Methods for making liposomes are well known in the art and are described elsewhere herein.

As used herein, the term “lipid” refers to a group of organic compounds that has lipophilic or amphipathic properties, including, but not limited to, fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids (lipochromes), and fatty acids. The term “lipid” encompasses both naturally occurring and synthetically produced lipids. “Lipophilic” refers to those organic compounds that dissolve in fats, oils, lipids, and non-polar solvents, such as organic solvents. Lipophilic compounds are sparingly soluble or insoluble in water. Thus, lipophilic compounds are hydrophobic. Amphipathic lipids, also referred to herein as “amphiphilic lipids” refer to a lipid molecule having both hydrophilic and hydrophobic characteristics. The hydrophobic group of an amphipathic lipid, as described in more detail immediately herein below, can be a long chain hydrocarbon group. The hydrophilic group of an amphipathic lipid can include a charged group, e.g., an anionic or a cationic group, or a polar, uncharged group. Amphipathic lipids can have multiple hydrophobic groups, multiple hydrophilic groups, and combinations thereof. Because of the presence of both a hydrophobic group and a hydrophilic group, amphipathic lipids can be soluble in water, and to some extent, in nonpolar organic solvents.

As used herein, “hydrophilic” is a physical property of a molecule that is capable of hydrogen bonding with a water (H₂O) molecule and is soluble in water and other polar solvents. The terms “hydrophilic” and “polar” can be used interchangeably. Hydrophilic characteristics derive from the presence of polar or charged groups, such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.

Conversely, the term “hydrophobic” is a physical property of a molecule that is repelled from a mass of water and can be referred to as “nonpolar,” or “apolar,” all of which are terms that can be used interchangeably with “hydrophobic.” Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).

Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and β-acyloxyacids, also are within the group designated as amphipathic lipids.

In some embodiments, the lipid vehicle of the delivery system comprises a cationic liposome. The term “cationic liposome” as used herein is intended to encompass any liposome as defined above which has a net positive charge or has a zeta potential of greater than 0 mV at physiological pH. The zeta potential or charge of the liposome can be measured using any method known to one of skill in the art. It should be noted that the liposome itself is the entity that is being determined as cationic, meaning that the liposome that has a measurable positive charge at physiological pH can, within an in vivo environment, become attached to other substances. These other substances can be negatively charged and thereby result in the formation of a structure that does not have a positive charge. After a delivery system comprising a cationic liposome is produced, molecules such as lipid-polyethylene glycol conjugates can be post-inserted into the bilayer of the liposome as described elsewhere herein, thus shielding the surface charge and reducing the zeta potential of the delivery system.

In some embodiments in which the lipid vehicle of the delivery system is a cationic liposome, the cationic liposome encapsulates an anionic bioactive compound (i.e., a bioactive compound that is negatively charged at physiological pH). In these embodiments, the positively charged cationic lipids comprising the cationic liposomes are physically associated with the negatively charged anionic bioactive compound by attraction between opposite molecular charges.

The cationic liposome comprises cationic amphipathic lipids. The positively charged cationic lipids comprising the cationic liposomes are physically associated with a negatively charged bioactive compound and/or polyanionic carrier macromolecule by attraction between opposite molecular charges. As used herein, “cationic lipid” encompasses any of a number of lipid species that carry a net positive charge at physiological pH, which can be determined using any method known to one of skill in the art. Such lipids include, but are not limited to the cationic lipids of formula (I) disclosed in International Application No. ______, entitled “Methods and Compositions Comprising Novel Cationic Lipids,” which was filed concurrently herewith, wherein formula (I) comprises:

wherein:

m and n are each independently integers from 1 to 8;

R₁ and R₂ are each independently —(CH₂)_(p)—CH₃, wherein p is an integer from 8 to 24;

R₃ and R₄ are each independently selected from the group consisting of H, alkyl, and substituted alkyl;

Q₁ and Q₂ are selected from the group consisting of:

wherein at least one of Q₁ or Q₂ is:

and pharmaceutically acceptable salts thereof.

In some embodiments, the cationic lipid of formula (I) comprises a lipid wherein m is 2, n is 4, p is 13, R₃ and R₄ are each methyl and the cationic lipid can be selected from the group consisting of:

referred to as N,N-di-myristoyl-N-methyl-N-2[N′—(N⁶-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DMGLA);

referred to as N,N-dimyristoyl-N-methyl-N-2[N²-guanidino-L-lysinyl]aminoethyl ammonium chloride; and

referred to as N,N-dimyristoyl-N-methyl-N-2[N′—(N²,N⁶-di-guanidino-L-lysinyl)]aminoethyl ammonium chloride.

In certain embodiments, the cationic lipid of formula (I) comprises a cationic lipid wherein m is 2, n is 3, p is 17, R₃ is H, R₄ is methyl,

and the lipid has the following chemical structure:

hereinafter referred to as N-methyl-N-(2-(arginoylamino) ethyl)-N,N-Di octadecyl aminium chloride or di stearoyl arginyl ammonium chloride] (DSAA).

In other embodiments, the cationic lipid of formula (I) comprises a cationic lipid wherein m is 2, n is 4, p is 17, R₃ and R₄ are each methyl,

and the cationic lipid of formula (I) has the following chemical structure:

and is referred to as N,N-di-stearoyl-N-methyl-N-2[N′—(N6-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DSGLA). In some of those embodiments wherein the bilayer comprises a cationic lipid of formula (I), the lipid has cytotoxic activity.

Other non-limiting examples of cationic lipids include N,N-di-myristoyl-N-methyl-N-2[N′—(N⁶-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DMGLA), N,N-dimyristoyl-N-methyl-N-2[N²-guanidino-L-lysinyl]aminoethyl ammonium chloride, N,N-dimyristoyl-N-methyl-N-2[N′—(N2,N6-di-guanidino-L-lysinyl)]aminoethyl ammonium chloride, N-methyl-N-(2-(arginoylamino) ethyl)-N,N-Di octadecyl-aminium chloride or di stearoyl arginyl ammonium chloride (DSAA), N,N-di-stearoyl-N-methyl-N-2[N′—(N6-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DSGLA). Non-limiting examples of other previously described cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); N-(2,3-dioleyloxy) propyl)-N,N,N-trimethylammonium chloride (“DOTMA”) or other N—(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl) cholesterol (“DC-Chol”) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”); 1,3-dioleoyl-3-trimethylammonium-propane, N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1 ammonium trifluoro-acetate (DOSPA); GAP-DLRIE; DMDHP; 3-β[⁴N—(¹N,⁸N-diguanidino spermidine)-carbamoyl]cholesterol (BGSC); 3-β[N,N-diguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N¹,N²,N³ Tetra-methyltetrapalmitylspermine (cellfectin); N-t-butyl-N′-tetradecyl-3-tetradecyl-aminopropion-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM) N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3 dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); 1,2 dioleoyl-3-(4′-trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl(4′ trimethylammonia)butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in International Application Publication No. WO 93/03709, which is herein incorporated by reference in its entirety; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES) or the cationic lipids disclosed in U.S. Pat. No. 5,283,185, which is herein incorporated by reference in its entirety; cholesteryl-3β-carboxyl-amido-ethylenetrimethylammonium iodide; 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide; cholesteryl-3-β-carboxyamidoethyleneamine; cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide; 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide; 2-(2-trimethylammonio)-ethylmethylamino ethyl-cholesteryl-3-β-oxysuccinate iodide; and 3-β-N-(polyethyleneimine)-carbamoylcholesterol.

The cationic liposome need not be comprised completely of cationic lipids, however, the cationic liposome comprises a sufficient amount of cationic lipids such that at physiological pH, the liposome has a positive charge. The cationic liposomes also can contain co-lipids that are negatively charged or neutral, so long as the net charge of the complexes formed is positive and/or the surface of the complex is positively charged at physiological pH. As used herein, a “co-lipid” refers to a non-cationic lipid, which includes neutral (uncharged), zwitterionic, or anionic lipids. The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. The term “anionic lipid” encompasses any of a number of lipid species that carry a net negative charge at physiological pH. Co-lipids can include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols, phospholipid-related materials, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidic acid, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, lysophosphatidylcholine, and dioctadecyldimethyl ammonium bromide and the like. Co-lipids also include polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to phospholipids or to ceramides, as described in U.S. Pat. No. 5,820,873, incorporated herein by reference.

In these embodiments, the ratio of cationic lipids to co-lipids is such that the overall charge of the resulting liposome is positive at physiological pH. For example, the cationic lipid is present in the liposome from about 10 mole % to about 100 mole % of total liposomal lipid, in some embodiments, from about 20 mole % to about 80 mole % and, in other embodiments, from about 20 mole % to about 60 mole %. The neutral lipid, when included in the liposome, can be present at a concentration of from about 0 mole % to about 90 mole % of the total liposomal lipid, in some embodiments from about 20 mole % to about 80 mole %, and in other embodiments, from about 40 mole % to about 80 mole %. The negatively charged lipid, when included in the liposome, can be present at a concentration ranging from about 0 mole % to about 49 mole % of the total liposomal lipid, and in certain embodiments, from about 0 mole % to about 40 mole %.

In some embodiments, the cationic liposomes of the presently disclosed polynucleotide delivery systems comprise the cationic lipid DOTAP and the neutral co-lipid cholesterol at a 1:1 molar ratio.

In some embodiments, the delivery system has a net positive charge, as described in U.S. Pat. No. 6,008,202, which is herein incorporated by reference in its entirety. By “net positive charge” is meant that the positive charges of the cationic liposome (and optionally, polycation, as described elsewhere herein) exceed the negative charge of an anionic bioactive compound (e.g., polynucleotide of interest) and/or a polyanionic carrier macromolecule. It is to be understood, however, that the present invention also encompasses delivery systems comprising a cationic liposome having a positively charged surface irrespective of whether the net charge of the complex is positive, neutral or even negative. The surface charge of a cationic liposome of a polynucleotide delivery system can be measured by the migration of the complex in an electric field by methods known to those in the art, such as by measuring zeta potential (Martin, Swarick, and Cammarata (1983) Physical Pharmacy & Physical Chemical Principles in the Pharmaceutical Sciences, 3rd ed. Lea and Febiger) or by the binding affinity of the polynucleotide delivery system complex to cell surfaces. Complexes exhibiting a positively charged surface have a greater binding affinity to cell surfaces than complexes having a neutral or negatively charged surface. Further, it is to be understood that the positively charged surface can be sterically shielded by the addition of non-ionic polar compounds, for example, polyethylene glycol, as described elsewhere herein.

In particular non-limiting embodiments, the delivery system has a charge ratio of positive to negative charge (+:−) of between about 0.5:1 and about 100:1, including but not limited to about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 40:1, or about 100:1. In some embodiments, the charge ratio is between about 0.5:1 and about 40:1, between about 0.5:1 and about 20:1, between about 0.5:1 and about 10:1, between about 2:1 and about 40:1, between about 2:1 and about 20:1, between about 2:1 and about 15:1, or between about 3:1 and about 9:1. In a specific non-limiting embodiment, the +:− charge ratio is about 5:1. The positive charge is imparted from the cationic liposome and in some embodiments, from the polycation and/or a cationic bioactive agent as well. The negative charge is imparted by an anionic bioactive agent (e.g., a polynucleotide of interest) and/or a polyanionic carrier macromolecule. Thus, in some embodiments, the liposome (+):polycation (+):anionic bioactive agent/polyanionic carrier macromolecule (−) charge ratio is about 4:1:1.

Methods for generating delivery systems comprising cationic liposomes as the lipid vehicle are known in the art, are presented elsewhere herein, and are reviewed in Li and Szoka (2007) Pharm. Res. 24:438-449.

B. Bioactive Compounds

The presently disclosed delivery systems comprise a bioactive compound. By “bioactive compound” is intended any agent that has an effect on a living cell, tissue, or organism, or an agent that can interact with a component (e.g., enzyme) of a living cell, tissue, or organism, including, but not limited to, polynucleotides, polypeptides, polysaccharides, organic and inorganic small molecules. The term “bioactive compound” encompasses both naturally occurring and synthetic bioactive compounds. The term “bioactive compound” can also refer to a detection or diagnostic agent that interacts with a biological molecule to provide a detectable readout that reflects a particular physiological or pathological event.

The bioactive compound of the delivery system can be a drug, including, but not limited to, antimicrobials, antibiotics, antimycobacterial, antifungals, antivirals, neoplastic agents, agents affecting the immune response, blood calcium regulators, agents useful in glucose regulation, anticoagulants, antithrombotics, antihyperlipidemic agents, cardiac drugs, thyromimetic and antithyroid drugs, adrenergics, antihypertensive agents, cholinergics, anticholinergics, antispasmodics, antiulcer agents, skeletal and smooth muscle relaxants, prostaglandins, general inhibitors of the allergic response, antihistamines, local anesthetics, analgesics, narcotic antagonists, antitussives, sedative-hypnotic agents, anticonvulsants, antipsychotics, anti-anxiety agents, antidepressant agents, anorexigenics, non-steroidal anti-inflammatory agents, steroidal anti-inflammatory agents, antioxidants, vaso-active agents, bone-active agents, antiarthritics, and diagnostic agents.

1. Polynucleotides of Interest

In those embodiments wherein the bioactive compound comprises a polynucleotide, the delivery system is referred to as a “polynucleotide delivery system” or “polynucleotide delivery system complex.” In some embodiments, a polynucleotide delivery system comprises a cationic liposome, a polynucleotide of interest, and a polyanionic carrier macromolecule, wherein the cationic liposome encapsulates the polynucleotide of interest and the polyanionic carrier macromolecule. The terms “encapsulate” and “entrap” are used herein interchangeably and refer to the incorporation or association of a substance or molecule (e.g., a polynucleotide) in or with a lipid vehicle. For example, in those embodiments in which the lipid vehicle is a liposome, the substance or molecule can be associated with the lipid bilayer (for example, if the substance or molecule is hydrophobic) or be present in the aqueous interior of the liposome (for example, if the substance or molecule is hydrophilic), or both. Polynucleotides are generally hydrophilic, anionic molecules and thus, are generally present within the aqueous interior of the liposome.

As used herein, the term “deliver” refers to the transfer of a substance or molecule (e.g., a polynucleotide) to a physiological site, tissue, or cell. This encompasses delivery to the intracellular portion of a cell or to the extracellular space. Delivery of a polynucleotide into the intracellular portion of a cell is also often referred to as “transfection.”

As used herein, the term “intracellular” or “intracellularly” has its ordinary meaning as understood in the art. In general, the space inside of a cell, which is encircled by a membrane, is defined as “intracellular” space. Similarly, as used herein, the term “extracellular” or “extracellularly” has its ordinary meaning as understood in the art. In general, the space outside of the cell membrane is defined as “extracellular” space.

The term “polynucleotide” is intended to encompass a singular nucleic acid, as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA), plasmid DNA (pDNA), or short interfering RNA (siRNA). A polynucleotide can be single-stranded or double-stranded, linear or circular. A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments or synthetic analogues thereof, present in a polynucleotide. The term “polynucleotide” can refer to an isolated polynucleotide, wherein an “isolated” nucleic acid or polynucleotide is a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. Examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. Isolated polynucleotides also can include isolated expression vectors, expression constructs, or populations thereof. “Polynucleotide” also can refer to amplified products of itself, as in a polymerase chain reaction. The “polynucleotide” can contain modified nucleic acids, such as phosphorothioate, phosphate, ring atom modified derivatives, and the like. The “polynucleotide” can be a naturally occurring polynucleotide (i.e., one existing in nature without human intervention), or a recombinant polynucleotide (i.e., one existing only with human intervention). While the terms “polynucleotide” and “oligonucleotide” both refer to a polymer of nucleotides, as used herein, an oligonucleotide is typically less than 100 nucleotides in length.

As used herein, the term “polynucleotide of interest” refers to a polynucleotide that is to be delivered to a cell to elicit a desired effect in the cell (e.g., a therapeutic effect, a change in gene expression). A polynucleotide of interest can be of any length and can include, but is not limited to a polynucleotide comprising a coding sequence for a polypeptide of interest or a polynucleotide comprising a silencing element. In certain embodiments, when the polynucleotide is expressed or introduced into a cell, the polynucleotide of interest or polypeptide encoded thereby has therapeutic activity.

By “therapeutic activity” when referring to a polynucleotide or a polypeptide encoded thereby is intended that the molecule is able to elicit a desired pharmacological or physiological effect when administered to a subject in need thereof.

a. Polynucleotides Encoding Polypeptides

In some embodiments, polynucleotide delivery systems comprise a polynucleotide of interest comprising a coding sequence for a polypeptide of interest.

For the purposes of the present invention, a “coding sequence for a polypeptide of interest” or “coding region for a polypeptide of interest” refers to the polynucleotide sequence that encodes that polypeptide. As used herein, the terms “encoding” or “encoded” when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to direct translation of the nucleotide sequence into a specified polypeptide. The information by which a polypeptide is encoded is specified by the use of codons. The “coding region” or “coding sequence” is the portion of the nucleic acid that consists of codons that can be translated into amino acids. Although a “stop codon” or “translational termination codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region. Likewise, a transcription initiation codon (ATG) may or may not be considered to be part of a coding region. Any sequences flanking the coding region, however, for example, promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not considered to be part of the coding region. In some embodiments, however, while not considered part of the coding region per se, these regulatory sequences and any other regulatory sequence, particularly signal sequences or sequences encoding a peptide tag, may be part of the polynucleotide sequence encoding the polypeptide of interest. Thus, a polynucleotide sequence encoding a polypeptide of interest comprises the coding sequence and optionally any sequences flanking the coding region that contribute to expression, secretion, and/or isolation of the polypeptide of interest.

The term “expression” has its meaning as understood in the art and refers to the process of converting genetic information encoded in a gene or a coding sequence into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of a polynucleotide (e.g., via the enzymatic action of an RNA polymerase), and for polypeptide-encoding polynucleotides, into a polypeptide through “translation” of mRNA. Thus, an “expression product” is, in general, an RNA transcribed from the gene (e.g., either pre- or post-processing) or polynucleotide or a polypeptide encoded by an RNA transcribed from the gene (e.g., either pre- or post-modification).

As used herein, the term “polypeptide” or “protein” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.

The term “polypeptide of interest” refers to a polypeptide that is to be delivered to a cell or is encoded by a polynucleotide that is to be delivered to a cell to elicit a desired effect in the cell (e.g., a therapeutic effect). The polypeptide of interest can be of any species and of any size. In certain embodiments, however, the protein or polypeptide of interest is a therapeutically useful protein or polypeptide. In some embodiments, the protein can be a mammalian protein, for example a human protein. In certain embodiments, the polynucleotide comprises a coding sequence for a tumor suppressor or a cytotoxin (e.g., diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), and the pertussis adenylate cyclase (CYA)).

The term “tumor suppressor” refers to a polypeptide or a gene that encodes a polypeptide that is capable of inhibiting the development, growth, or progression of cancer. Tumor suppressor polypeptides include those proteins that regulate cellular proliferation or responses to cellular and genomic damage, or induce apoptosis. Non-limiting examples of tumor suppressor genes include p53, p110Rb, and p72. Thus, in some embodiments, the polynucleotide delivery systems of the present invention comprise a polynucleotide of interest comprising a coding sequence for a tumor suppressor.

Extensive sequence information required for molecular genetics and genetic engineering techniques is widely publicly available. Access to complete nucleotide sequences of mammalian, as well as human, genes, cDNA sequences, amino acid sequences and genomes can be obtained from GenBank at the website www.ncbi.nlm.nih.gov/Entrez. Additional information can also be obtained from GeneCards, an electronic encyclopedia integrating information about genes and their products and biomedical applications from the Weizmann Institute of Science Genome and Bioinformatics (bioinformatics.weizmann.ac.il/cards), nucleotide sequence information can be also obtained from the EMBL Nucleotide Sequence Database (www.ebi.ac.uk/embl) or the DNA Databank or Japan (DDBJ, www.ddbi.nig.ac.jp). Additional sites for information on amino acid sequences include Georgetown's protein information resource website (www.pir.georgetown.edu) and Swiss-Prot (au.expasy.org/sprot/sprot-top.html).

b. Silencing Elements

In some embodiments, the polynucleotide of interest of the polynucleotide delivery systems of the invention comprises a silencing element, wherein expression or introduction of the silencing element into a cell reduces the expression of a target polynucleotide or polypeptide encoded thereby.

The terms “introduction” or “introduce” when referring to a polynucleotide or silencing element refers to the presentation of the polynucleotide or silencing element to a cell in such a manner that the polynucleotide or silencing element gains access to the intracellular region of the cell.

As used herein, the term “silencing element” refers to a polynucleotide, which when expressed or introduced into a cell is capable of reducing or eliminating the level of expression of a target polynucleotide sequence or the polypeptide encoded thereby. The silencing element can comprise or encode an antisense oligonucleotide or an interfering RNA (RNAi). The term “interfering RNA” or “RNAi” refers to any RNA molecule which can enter an RNAi pathway and thereby reduce the expression of a target polynucleotide of interest. The RNAi pathway features the Dicer nuclease enzyme and RNA-induced silencing complexes (RISC) that function to degrade or block the translation of a target mRNA. RNAi is distinct from antisense oligonucleotides that function through “antisense” mechanisms that typically involve inhibition of a target transcript by a single-stranded oligonucleotide through an RNase H-mediated pathway. See, Crooke (ed.) (2001) “Antisense Drug Technology: Principles, Strategies, and Applications” (1st ed), Marcel Dekker; ISBN: 0824705661; 1st edition.

As used herein, a “target polynucleotide” comprises any polynucleotide sequence that one desires to decrease the level of expression. By “reduces” or “reducing” the expression level of a polynucleotide or a polypeptide encoded thereby is intended to mean, the level of the polynucleotide or the encoded polypeptide is statistically lower than the target polynucleotide level or encoded polypeptide level in an appropriate control which is not exposed to the silencing element. In particular embodiments, reducing the target polynucleotide level and/or the encoded polypeptide level according to the presently disclosed subject matter results in less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the target polynucleotide level, or the level of the polypeptide encoded thereby in an appropriate control. Methods to assay for the level of the RNA transcript, the level of the encoded polypeptide, or the activity of the polynucleotide or polypeptide are discussed elsewhere herein.

In some embodiments, the target polynucleotide is an oncogene or a proto-oncogene. The term “oncogene” is used herein in accordance with its art-accepted meaning to refer to those polynucleotide sequences that encode a gene product that contributes to cancer initiation or progression. The term “oncogene” encompasses proto-oncogenes, which are genes that do not contribute to carcinogenesis under normal circumstances, but that have been mutated, overexpressed, or activated in such a manner as to function as an oncogene. Non-limiting examples of oncogenes include growth factors or mitogens (e.g., c-Sis), receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), HER2/neu), cytoplasmic tyrosine kinases (e.g., src, Ab1), cytoplasmic serine/threonine kinases (e.g., raf kinase, cyclin-dependent kinases), regulatory GTPases (e.g., ras), and transcription factors (e.g., myc). In some embodiments, the target polynucleotide is EGFR.

The term “complementary” is used herein in accordance with its art-accepted meaning to refer to the capacity for precise pairing via hydrogen bonds (e.g., Watson-Crick base pairing or Hoogsteen base pairing) between two nucleosides, nucleotides or nucleic acids, and the like. For example, if a nucleotide at a certain position of a first nucleic acid is capable of stably hydrogen bonding with a nucleotide located opposite to that nucleotide in a second nucleic acid, when the nucleic acids are aligned in opposite 5′ to 3′ orientation (i.e., in anti-parallel orientation), then the nucleic acids are considered to be complementary at that position (where position may be defined relative to either end of either nucleic acid, generally with respect to a 5′ end). The nucleotides located opposite one another can be referred to as a “base pair.” A complementary base pair contains two complementary nucleotides, e.g., A and U, A and T, G and C, and the like, whereas a noncomplementary base pair contains two noncomplementary nucleotides (also referred to as a mismatch). Two polynucleotides are said to be complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that hydrogen bond with each other, i.e., a sufficient number of base pairs are complementary.

As used herein, the term “gene” has its meaning as understood in the art. In general, a gene is taken to include gene regulatory sequences (e.g., promoters, enhancers, and the like) and/or intron sequences, in addition to coding sequences (open reading frames). It will further be appreciated that definitions of “gene” include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules, or precursors thereof, such as microRNA or siRNA precursors, tRNAs, and the like.

The term “hybridize” as used herein refers to the interaction between two complementary nucleic acid sequences in which the two sequences remain associated with one another under appropriate conditions.

A silencing element can comprise the interfering RNA or antisense oligonucleotide, a precursor to the interfering RNA or antisense oligonucleotide, a template for the transcription of an interfering RNA or antisense oligonucleotide, or a template for the transcription of a precursor interfering RNA or antisense oligonucleotide, wherein the precursor is processed within the cell to produce an interfering RNA or antisense oligonucleotide. Thus, for example, a dsRNA silencing element includes a dsRNA molecule, a transcript or polyribonucleotide capable of forming a dsRNA, more than one transcript or polyribonucleotide capable of forming a dsRNA, a DNA encoding a dsRNA molecule, or a DNA encoding one strand of a dsRNA molecule. When the silencing element comprises a DNA molecule encoding an interfering RNA, it is recognized that the DNA can be transiently expressed in a cell or stably incorporated into the genome of the cell. Such methods are discussed in further detail elsewhere herein.

The silencing element can reduce or eliminate the expression level of a target polynucleotide or encoded polypeptide by influencing the level of the target RNA transcript, by influencing translation, or by influencing expression at the pre-transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). See, for example, Verdel et al. (2004) Science 303:672-676; Pal-Bhadra et al. (2004) Science 303:669-672; Allshire (2002) Science 297:1818-1819; Volpe et al. (2002) Science 297:1833-1837; Jenuwein (2002) Science 297:2215-2218; and Hall et al. (2002) Science 297:2232-2237. Methods to assay for functional interfering RNA that are capable of reducing or eliminating the level of a sequence of interest are disclosed elsewhere herein.

Any region of the target polynucleotide can be used to design a domain of the silencing element that shares sufficient sequence identity to allow for the silencing element to decrease the level of the target polynucleotide or encoded polypeptide. For instance, the silencing element can be designed to share sequence identity to the 5′ untranslated region of the target polynucleotide(s), the 3′ untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof.

The ability of a silencing element to reduce the level of the target polynucleotide can be assessed directly by measuring the amount of the target transcript using, for example, Northern blots, nuclease protection assays, reverse transcription (RT)-PCR, real-time RT-PCR, microarray analysis, and the like. Alternatively, the ability of the silencing element to reduce the level of the target polynucleotide can be measured directly using a variety of affinity-based approaches (e.g., using a ligand or antibody that specifically binds to the target polypeptide) including, but not limited to, Western blots, immunoassays, ELISA, flow cytometry, protein microarrays, and the like. In still other methods, the ability of the silencing element to reduce the level of the target polynucleotide can be assessed indirectly, e.g., by measuring a functional activity of the polypeptide encoded by the transcript or by measuring a signal produced by the polypeptide encoded by the transcript.

Various types of silencing elements are discussed in further detail below.

i. Double Stranded RNA Silencing Elements

In one embodiment, the silencing element comprises or encodes a double stranded RNA molecule. As used herein, a “double stranded RNA” or “dsRNA” refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. Accordingly, as used herein, the term “dsRNA” is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, for example, small RNA (sRNA), short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), and others. See, for example, Meister and Tuschl (2004) Nature 431:343-349 and Bonetta et al. (2004) Nature Methods 1:79-86.

In specific embodiments, at least one strand of the duplex or double-stranded region of the dsRNA shares sufficient sequence identity or sequence complementarity to the target polynucleotide to allow for the dsRNA to reduce the level of expression of the target polynucleotide or encoded polypeptide. As used herein, the strand that is complementary to the target polynucleotide is the “antisense strand,” and the strand homologous to the target polynucleotide is the “sense strand.”

In one embodiment, the dsRNA comprises a hairpin RNA. A hairpin RNA comprises an RNA molecule that is capable of folding back onto itself to form a double stranded structure. Multiple structures can be employed as hairpin elements. For example, the hairpin RNA molecule that hybridizes with itself to form a hairpin structure can comprise a single-stranded loop region and a base-paired stem. The base-paired stem region can comprise a sense sequence corresponding to all or part of the target polynucleotide and further comprises an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the silencing element can determine the specificity of the silencing. See, for example, Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990, herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga et al. (2003) Mol. Biol. Rep. 30:135-140, herein incorporated by reference.

ii. siRNA Silencing Elements

A “short interfering RNA” or “siRNA” comprises an RNA duplex (double-stranded region) and can further comprise one or two single-stranded overhangs, e.g., 3′ or 5′ overhangs. The duplex can be approximately 19 base pairs (bp) long, although lengths between 17 and 29 nucleotides, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 nucleotides, can be used. An siRNA can be formed from two RNA molecules that hybridize together or can alternatively be generated from a single RNA molecule that includes a self-hybridizing portion. The duplex portion of an siRNA can include one or more bulges containing one or more unpaired and/or mismatched nucleotides in one or both strands of the duplex or can contain one or more noncomplementary nucleotide pairs. One strand of an siRNA (referred to herein as the antisense strand) includes a portion that hybridizes with a target transcript. In certain embodiments, one strand of the siRNA (the antisense strand) is precisely complementary with a region of the target transcript over at least about 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or more meaning that the siRNA antisense strand hybridizes to the target transcript without a single mismatch (i.e., without a single noncomplementary base pair) over that length. In other embodiments, one or more mismatches between the siRNA antisense strand and the targeted portion of the target transcript can exist. In embodiments in which perfect complementarity is not achieved, any mismatches between the siRNA antisense strand and the target transcript can be located at or near the 3′ end of the siRNA antisense strand. For example, in certain embodiments, nucleotides 1-9, 2-9, 2-10, and/or 1-10 of the antisense strand are perfectly complementary to the target.

Considerations for the design of effective siRNA molecules are discussed in McManus et al. (2002) Nature Reviews Genetics 3: 737-747 and in Dykxhoorn et al. (2003) Nature Reviews Molecular Cell Biology 4: 457-467. Such considerations include the base composition of the siRNA, the position of the portion of the target transcript that is complementary to the antisense strand of the siRNA relative to the 5′ and 3′ ends of the transcript, and the like. A variety of computer programs also are available to assist with selection of siRNA sequences, e.g., from Ambion (web site having URL www.ambion.com), at the web site having the URL www.sinc.sunysb.edu/Stu/shilin/rnai.html. Additional design considerations that also can be employed are described in Semizarov et al. Proc. Natl. Acad. Sci. 100: 6347-6352.

iii. Short Hairpin RNA Silencing Elements

The term “short hairpin RNA” or “shRNA” refers to an RNA molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a double-stranded (duplex) structure sufficiently long to mediate RNAi (generally between approximately 17 and 29 nucleotides in length, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 nucleotides in length, and in some embodiments, typically at least 19 base pairs in length), and at least one single-stranded portion, typically between approximately 1 and 20 or 1 to 10 nucleotides in length that forms a loop connecting the two nucleotides that form the base pair at one end of the duplex portion. The duplex portion can, but does not require, one or more bulges consisting of one or more unpaired nucleotides. In specific embodiments, the shRNAs comprise a 3′ overhang. Thus, shRNAs are precursors of siRNAs and are, in general, similarly capable of inhibiting expression of a target transcript.

In particular, RNA molecules having a hairpin (stem-loop) structure can be processed intracellularly by Dicer to yield an siRNA structure referred to as short hairpin RNAs (shRNAs), which contain two complementary regions that hybridize to one another (self-hybridize) to form a double-stranded (duplex) region referred to as a stem, a single-stranded loop connecting the nucleotides that form the base pair at one end of the duplex, and optionally an overhang, e.g., a 3′ overhang. The stem can comprise about 19, 20, or 21 bp long, though shorter and longer stems (e.g., up to about 29 nt) also can be used. The loop can comprise about 1-20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nt, about 4-10, or about 6-9 nt. The overhang, if present, can comprise approximately 1-20 nt or approximately 2-10 nt. The loop can be located at either the 5′ or 3′ end of the region that is complementary to the target transcript whose inhibition is desired (i.e., the antisense portion of the shRNA).

Although shRNAs contain a single RNA molecule that self-hybridizes, it will be appreciated that the resulting duplex structure can be considered to comprise sense and antisense strands or portions relative to the target mRNA and can thus be considered to be double-stranded. It will therefore be convenient herein to refer to sense and antisense strands, or sense and antisense portions, of an shRNA, where the antisense strand or portion is that segment of the molecule that forms or is capable of forming a duplex with and is complementary to the targeted portion of the target polynucleotide, and the sense strand or portion is that segment of the molecule that forms or is capable of forming a duplex with the antisense strand or portion and is substantially identical in sequence to the targeted portion of the target transcript. In general, considerations for selection of the sequence of the antisense strand of an shRNA molecule are similar to those for selection of the sequence of the antisense strand of an siRNA molecule that targets the same transcript.

iv. MicroRNA Silencing Elements

In one embodiment, the silencing element comprises or encodes an miRNA or an miRNA precursor. “MicroRNAs” or “miRNAs” are regulatory agents comprising about 19 ribonucleotides which are highly efficient at inhibiting the expression of target polynucleotides. See, for example, Saetrom et al. (2006) Oligonucleotides 16:115-144, Wang et al. (2006) Mol. Cell 22:553-60, Davis et al. (2006) Nucleic Acid Research 34:2294-304, Pasquinelli (2006) Dev. Cell 10:419-24, all of which are herein incorporated by reference. For miRNA interference, the silencing element can be designed to express a dsRNA molecule that forms a hairpin structure containing a 19-nucleotide sequence that is complementary to the target polynucleotide of interest. The miRNA can be synthetically made, or transcribed as a longer RNA which is subsequently cleaved to produce the active miRNA. Specifically, the miRNA can comprise 19 nucleotides of the sequence having homology to a target polynucleotide in sense orientation and 19 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence.

It is recognized that various forms of an miRNA can be transcribed including, for example, the primary transcript (termed the “pri-miRNA”) which is processed through various nucleolytic steps to a shorter precursor miRNA (termed the “pre-miRNA”); the pre-miRNA; or the final (mature) miRNA, which is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) and miRNA*. The pre-miRNA is a substrate for a form of dicer that removes the miRNA/miRNA* duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (McManus et al. (2002) RNA 8:842-50). In specific embodiments, 2-8 nucleotides of the miRNA are perfectly complementary to the target. A large number of endogenous human miRNAs have been identified. For structures of a number of endogenous miRNA precursors from various organisms, see Lagos-Quintana et al. (2003) RNA 9(2):175-9; see also Bartel (2004) Cell 116:281-297.

A miRNA or miRNA precursor can share at least about 80%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with the target transcript for a stretch of at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In specific embodiments, the region of precise sequence complementarity is interrupted by a bulge. See, Ruvkun (2001) Science 294: 797-799, Zeng et al. (2002) Molecular Cell 9:1-20, and Mourelatos et al. (2002) Genes Dev 16:720-728.

v. Antisense Silencing Elements

In some embodiments, the silencing element comprises or encodes an antisense oligonucleotide. An “antisense oligonucleotide” is a single-stranded nucleic acid sequence that is wholly or partially complementary to a target polynucleotide, and can be DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart).

The antisense oligonucleotides of this invention are designed to be hybridizable with target RNA (e.g., mRNA) or DNA. For example, an oligonucleotide (e.g., DNA oligonucleotide) that hybridizes to a mRNA molecule can be used to target the mRNA for RnaseH digestion. Alternatively, an oligonucleotide that hybridizes to the translation initiation site of an mRNA molecule can be used to prevent translation of the mRNA. In another approach, oligonucleotides that bind to double-stranded DNA can be administered. Such oligonucleotides can form a triplex construct and inhibit the transcription of the DNA. Triple helix pairing prevents the double helix from opening sufficiently to allow the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described (see, e.g., J. E. Gee et al., 1994, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Such oligonucleotides of the invention can be constructed using the base-pairing rules of triple helix formation and the nucleotide sequences of the target genes.

As non-limiting examples, antisense oligonucleotides can be targeted to hybridize to the following regions: mRNA cap region; translation initiation site; translational termination site; transcription initiation site; transcription termination site; polyadenylation signal; 3′ untranslated region; 5′ untranslated region; 5′ coding region; mid coding region; and 3′ coding region. In some embodiments, the complementary oligonucleotide is designed to hybridize to the most unique 5′ sequence of a gene, including any of about 15-35 nucleotides spanning the 5′ coding sequence.

Accordingly, the antisense oligonucleotides in accordance with this invention can comprise from about 10 to about 100 nucleotides, including, but not limited to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 nucleotides.

Antisense nucleic acids can be produced by standard techniques (see, for example, Shewmaker et al., U.S. Pat. No. 5,107,065). Appropriate oligonucleotides can be designed using OLIGO software (Molecular Biology Insights, Inc., Cascade, Colo.; http://www.oligo.net).

vi. Preparing Silencing Elements

Those of ordinary skill in the art will readily appreciate that a silencing element can be prepared according to any available technique including, but not limited to, chemical synthesis, enzymatic or chemical cleavage in vivo or in vitro, template transcription in vivo or in vitro, or combinations of the foregoing.

c. Recombinant Expression Vectors and Host Cells

As discussed above, the silencing elements employed in the methods and compositions of the invention can comprise a DNA molecule which when transcribed produces an interfering RNA or a precursor thereof, or an antisense oligonucleotide. In such embodiments, the DNA molecule encoding the silencing element is found in an expression cassette. In addition, polynucleotides that comprise a coding sequence for a polypeptide of interest are found in an expression cassette.

The expression cassette comprises one or more regulatory sequences, selected on the basis of the cells to be used for expression, operably linked to a polynucleotide encoding the silencing element or polypeptide of interest. “Operably linked” is intended to mean that the nucleotide sequence of interest (i.e., a DNA encoding a silencing element or a coding sequence for a polypeptide of interest) is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a cell when the expression cassette or vector is introduced into a cell). “Regulatory sequences” include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression cassette can depend on such factors as the choice of the host cell to be transformed, the level of expression of the silencing element or polypeptide of interest desired, and the like. Such expression cassettes typically include one or more appropriately positioned sites for restriction enzymes, to facilitate introduction of the nucleic acid into a vector.

It will further be appreciated that appropriate promoter and/or regulatory elements can readily be selected to allow expression of the relevant transcription units/silencing elements in the cell of interest. In certain embodiments, the promoter utilized to direct intracellular expression of a silencing element is a promoter for RNA polymerase III (Pol III). References discussing various Pol III promoters, include, for example, Yu et al. (2002) Proc. Natl. Acad. Sci. 99(9), 6047-6052; Sui et al. (2002) Proc. Natl. Acad. Sci. 99(8), 5515-5520 (2002); Paddison et al. (2002) Genes and Dev. 16, 948-958; Brummelkamp et al. (2002) Science 296, 550-553; Miyagashi (2002) Biotech. 20, 497-500; Paul et al. (2002) Nat. Biotech. 20, 505-508; Tuschl et al. (2002) Nat. Biotech. 20, 446-448. According to other embodiments, a promoter for RNA polymerase I, e.g., a tRNA promoter, can be used. See McCown et al. (2003) Virology 313(2):514-24; Kawasaki (2003) Nucleic Acids Res. 31 (2):700-7. In some embodiments in which the polynucleotide comprises a coding sequence for a polypeptide of interest, a promoter for RNA polymerase II can be used.

The regulatory sequences can also be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

In vitro transcription can be performed using a variety of available systems including the T7, SP6, and T3 promoter/polymerase systems (e.g., those available commercially from Promega, Clontech, New England Biolabs, and the like). Vectors including the T7, SP6, or T3 promoter are well known in the art and can readily be modified to direct transcription of silencing elements. When silencing elements are synthesized in vitro, the strands can be allowed to hybridize before introducing into a cell or before administration to a subject. As noted above, silencing elements can be delivered or introduced into a cell as a single RNA molecule including self-complementary portions (e.g., an shRNA that can be processed intracellularly to yield an siRNA), or as two strands hybridized to one another. In other embodiments, the silencing elements employed are transcribed in vivo. As discussed elsewhere herein, regardless of if the silencing element is transcribed in vivo or in vitro, in either scenario, a primary transcript can be produced which can then be processed (e.g., by one or more cellular enzymes) to generate the interfering RNA that accomplishes gene inhibition.

In those embodiments in which the silencing element is an interfering RNA, the interfering RNA can be generated by transcription from a promoter, either in vitro or in vivo. For instance, a construct can be provided containing two separate transcribable regions, each of which generates a 21 nt transcript containing a 19 nt region complementary with the other. Alternatively, a single construct can be utilized that contains opposing promoters and terminators positioned so that two different transcripts, each of which is at least partly complementary to the other, are generated. Alternatively, an RNA-inducing agent can be generated as a single transcript, for example by transcription of a single transcription unit encoding self complementary regions. A template is employed that includes first and second complementary regions, and optionally includes a loop region connecting the portions. Such a template can be utilized for in vitro transcription or in vivo transcription, with appropriate selection of promoter and, optionally, other regulatory elements, e.g., a terminator.

2. Polypeptides of Interest

In some embodiments, the presently disclosed delivery systems comprise a lipid vehicle encapsulating a polypeptide of interest that is to be delivered to a cell. The delivery systems disclosed herein are capable of introducing a polypeptide into the intracellular region of a cell. Importantly, targeted delivery systems comprising a lipid vehicle encapsulating a polypeptide are capable of specifically delivering a given polypeptide into a cell.

In some embodiments, the delivery system capable of delivering a polypeptide of interest into a cell comprises a cationic liposome encapsulating the polypeptide. In certain embodiments, the cationic liposome comprises the cationic lipid DOTAP and the co-lipid cholesterol in a 1:1 molar ratio. In some of these embodiments, the polypeptide that is delivered into the cell comprises an anionic polypeptide. As used herein, an “anionic polypeptide” is a polypeptide as described herein that has a net negative charge at physiological pH. The anionic polypeptide can comprise at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid residues that have a negative charge at physiological pH. These include aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q). In particular embodiments, the polypeptide of interest is acetylated at the amino and/or carboxyl termini to enhance the negative charge of the polypeptide.

In some embodiments, the delivery system further comprises a polycation, as described elsewhere herein. In other embodiments, the delivery system comprises a polyanionic carrier macromolecule, such as heparin sulfate. In particular embodiments, the delivery system comprises a LPH nanoparticle, which comprises a cationic liposome encapsulating a polypeptide of interest, heparin sulfate, and a polycation (e.g., protamine). In some of these embodiments, the polypeptide comprises an anionic polypeptide. In certain embodiments, the delivery system comprises a cationic liposome encapsulating an anionic polypeptide, and optionally a polyanionic carrier macromolecule (e.g., heparin sulfate, calf thymus DNA, hyaluronic acid) and a polycation (e.g., protamine) and the surface charge of the cationic liposome is shielded by the post-insertion of lipid-polyethylene glycol conjugates into the lipid bilayer of the liposome. In some of these embodiments, the delivery system is a stealth delivery system. To provide specific targeting to a targeted cell or tissue, the outer leaflet of the lipid bilayer of the liposome of the delivery system can further comprise a targeting ligand.

In some embodiments, the delivery systems comprising a polypeptide of interest comprise polypeptides of interest having at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more amino acid residues. In some embodiments, the polypeptide of interest comprises 9 amino acid residues, which can also be referred to as a nonapeptide. In other specific embodiments, the polypeptide of interest comprises 16 amino acid residues. The polypeptide of interest that is delivered to a cell using the delivery systems disclosed herein can have a molecular weight from about 200 Daltons to about 50,000 Daltons, including but not limited to, about 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, and 50,000 Daltons. In particular embodiments, wherein the delivery system comprises a lipid vehicle encapsulating the polypeptide of interest, the delivery system is capable of delivering between about 1 and about 2×10¹⁶ molecules of the polypeptide of interest in a single lipid vehicle, including but not limited to about 1, 10, 100, 500, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, and 2×10¹⁶ molecules.

In some embodiments, the polypeptide of interest is capable of blocking an essential intracellular signaling event upon delivery to a cell and is cytotoxic to the cell. In some of these embodiments, the polypeptide of interest comprises an amino acid sequence that mimics the catalytic domain of an enzyme that functions in an essential signaling pathway in the cell. A non-limiting example of such an enzyme is the epidermal growth factor receptor (EGFR) tyrosine kinase. Upon activation, EGFR autophosphorylates at tyrosine (Y) 845 and phosphorylates many downstream targets, including STAT proteins, such as STAT5b, which is involved in cell cycle, survival, and proliferation (Matsumura et al. (1999) EMBO J 18:1367-1377; Quelle et al. (1996) Mol Cell Biol 16:1622-1631; Matsumura et al. (1997) Mol Cell Biol 17:2933-2943). In some of the embodiments wherein the polypeptide of interest that is to be delivered to a cell and mimic the catalytic domain of EGFR has the sequence of EEEE(pY)FELV, wherein pY represents phosphotyrosine). This nonapeptide is referred to herein as the EV peptide and its sequence is set forth in SEQ ID NO: 3. As demonstrated elsewhere herein, delivery of the EV peptide to cells resulted in a growth arrest and induction of apoptosis (see Experimental Example 6).

In other embodiments wherein the delivery system comprises a polypeptide of interest, the polypeptide of interest is used as an imaging or diagnostic tool and is referred to herein as an imaging polypeptide. In some of these embodiments, the polypeptide comprises at least one caspase 3 recognition motif, which is described in Nicholson et al. (1995) Nature 376(6535):37-43, which is herein incorporated by reference in its entirety. Upon activation, caspase 3 binds to and cleaves peptides with the amino acid sequence of DEVD (set forth in SEQ ID NO: 5), which is referred to herein as the caspase 3 recognition motif. In some embodiments, the polypeptide comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more caspase 3 recognition motifs. In particular embodiments, the polypeptide of interest comprises three caspase 3 recognition motifs.

In order to detect cleavage of the peptide by caspase 3, a donor fluorophore can be conjugated to a first amino acid in the polypeptide and an acceptor fluorophore can be conjugated to a second amino acid and fluorescene resonance energy transfer (FRET) between the two fluorophores is expected to occur when the two fluorophores are in close proximity to one another. As used herein when referring to FRET technology, a “donor fluorophore” is the fluorophore that is generally excited by the application of an external light source within the excitation spectrum of the donor fluorophore, and once excited, the donor fluorophore emits light at another wavelength spectrum, referred to as the emission spectrum. When in close proximity to the donor fluorophore, fluorescence resonance energy transfer occurs between the donor fluorophore and the acceptor fluorophore, wherein the “acceptor fluorophore” is excited by the fluorescent light emitted by the donor fluorophore and the acceptor fluorophore emits light of a different wavelength. Thus, in order for FRET to occur between the two fluorophores, the emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor fluorophore. In some embodiments, in order for FRET to occur, the distance between the donor fluorophore and the acceptor fluorophore is less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, less than about 9 nm, less than about 8 nm, less than about 7 nm, less than about 6 nm, less than about 5 nm, less than about 4 nm, less than about 3 nm, less than about 2 nm, less than about 1 nm, or less. Therefore, in some embodiments, the donor fluorophore is conjugated to a first amino acid residue in the polypeptide of interest and the acceptor fluorophore is conjugated to a second amino acid residue with about 1 to about 50 amino acid residues in between the first and the second amino acid residue, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acid residues.

In some embodiments, the polypeptide of interest comprising at least one caspase 3 motif comprises the sequence of KDEVDCDEVDKDEVDC (set forth in SEQ ID NO: 6), wherein a first fluorophore is conjugated to the thiol group of the cysteines (C) and a second fluorophore is conjugated to the amino group of the lysines (K). In some embodiments, the first fluorophore (conjugated to cysteine) is the donor fluorophore and the second fluorophore (conjugated to lysine) is the acceptor fluorophore. In other embodiments, the first fluorophore (conjugated to cysteine) is the acceptor fluorophore and the second fluorophore (conjugated to lysine) is the donor fluorophore. Non-limiting examples of fluorophores that can be used for the presently disclosed compositions include Cy3, Cy5, Cy5.5, Cy7, Alexa488, Alexa555, FITC, and rhodamine (TRITC). It is to be noted that the selection of the donor fluorophore depends on the excitation and emission spectra of the acceptor fluorophore and vice versa. Frequently used fluorophore pairs for FRET include but are not limited to, Cy3 and Cy5, Alexa488 and Alexa555, Alexa488 and Cy3, and FITC and rhodamine. In certain embodiments, the donor fluorophore comprises Cy5.5 and the acceptor fluorophore comprises Cy7. In some of these embodiments, Cy5.5 is conjugated to at least one cysteine residue of SEQ ID NO: 6 through the thiol group of the cysteine and Cy7 is conjugated to at least one lysine residue of SEQ ID NO: 6 through the amino group of the lysine. In some embodiments wherein the amino acid sequence of the polypeptide of interest comprises the sequence set forth in SEQ ID NO: 6, the amino terminus of the polypeptide is acetylated, contributing to the negative charge of the polypeptide.

In some embodiments wherein the polypeptide of interest comprises a polypeptide comprising at least one caspase 3 recognition motif and a donor and acceptor fluorophore conjugated to a first and second amino acid residue of the polypeptide at a distance sufficient for FRET to occur between the two fluorophores, the delivery system can comprise the polypeptide of interest and a means for delivering the polypeptide into a cell (so that the polypeptide is placed in the intracellular region of the cell). Therefore, the delivery system can comprise any means for intracellular delivery of a polypeptide, which can include fusion polypeptide comprising a protein transduction domain, a polypeptide that possesses the ability to transverse biological membranes efficiently in a process termed protein transduction, or a lipid vehicle that encapsulates the polypeptide of interest. PTDs can translocate the cell membrane freely in a way that is receptor-, or transporter-independent, non-saturable, and consumes no energy. The PTD can cross the cell membrane within less than one hour. The most widely recognized protein transduction domain is that of the HIV TAT protein (Green and Lowenstein (1988) Cell 55:1179-1188; Frankel and Pabo (1988) Cell 55:1189-1913). The minimal protein transduction domain of the TAT protein is amino acid residues 49-57 of the protein. Other proteins have been shown to comprise protein transduction domains as well (Elliott and O'Hare (1997) Cell 88:223-233; Joliot et al. (1991) Proc Natl Acad Sci USA 88:1864-1868; Chatelin et al. (1996) Mech Dev 55:111-117). A polypeptide of interest can be delivered into a cell through the fusion of at least one protein transduction domain (also referred to herein as a cell-penetrating polypeptide) at the amino and/or carboxyl termini of the polypeptide of interest or cross-linking of the PTD to the polypeptide of interest. Non-limiting examples of suitable PTD sequences can be found, for example, in U.S. Pat. Nos. 6,835,810 and 7,306,944, each of which is herein incorporated by reference in their entireties. The fundamental requirements for the creation, isolation, and utilization of TAT-fusion proteins to transducer mammalian cells were described in Becker-Hapak et al. (2001) Methods 24:247-256, which is herein incorporated by reference in its entirety. In some embodiments, the PTD that is fused to a polypeptide of interest comprises a relatively small polypeptide that is rich in arginine and/or lysine residues. In particular embodiments, the PTD has a length of about 5 amino acid residues to about 100 amino acid residues, including but not limited to, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, and 100 amino acid residues, with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues being an arginine or a lysine residue.

In other embodiments, the means for delivering the polypeptide of interest comprising at least one caspase 3 recognition motif and an acceptor fluorophore and donor fluorophore at a sufficient distance for FRET to occur comprises a lipid vehicle, such as a cationic liposome, that encapsulates the polypeptide. In these embodiments, the polypeptide can have an overall negative charge (i.e., an anionic polypeptide). In some of these embodiments, the delivery system comprises a cationic liposome encapsulating the polypeptide of interest, a polyanionic carrier macromolecule (e.g., heparin sulfate), and a polycation (e.g., protamine sulfate). The surface charge of the liposome can be fully or partially shielded through the insertion of lipid-PEG conjugates into the outer leaflet of the lipid bilayer of the liposome. Further, the delivery system can be targeted to particular cells through the association of a targeting ligand with the liposome (e.g., through the post-insertion of lipid-targeting ligands or lipid-PEG-targeting ligand conjugates into the outer leaflet of the lipid bilayer of the liposome). In some of these embodiments, the polypeptide of interest has the amino acid sequence set forth in SEQ ID NO: 6, optionally acetylated at the amino terminus.

In certain embodiments wherein the delivery system comprises a polypeptide of interest comprising at least one caspase 3 recognition motif and conjugated to a donor fluorophore and an acceptor fluorophore (i.e., an imaging polypeptide), the delivery system further comprises a cytotoxic bioactive compound. Thus, the delivery system can function as a theranostic when delivered to a subject that would benefit from the cytotoxic properties of the bioactive compound (for example, a subject with cancer). As used herein, the term “theranostic” refers to the ability of a compound or complex to exert a therapeutic effect while also diagnosing the presence or progression of a particular disease or unwanted condition or monitoring the efficacy of a therapeutic. For example, the theranostic delivery system comprising an imaging polypeptide comprising at least one caspase 3 recognition motif and a cytotoxic bioactive compound can be used to inhibit cell growth and induce apoptosis while simultaneously providing an image of those cells undergoing apoptosis in real time. In some of these embodiments, the cytotoxic bioactive compound comprises the EV peptide set forth in SEQ ID NO: 3.

The presently disclosed subject matter also provides an isolated polypeptide, wherein the polypeptide comprises at least one caspase 3 recognition motif and is conjugated to a donor fluorophore through a first amino acid and an acceptor fluorophore through a second amino acid (i.e., an imaging polypeptide). In particular embodiments, the isolated polypeptide has the sequence set forth in SEQ ID NO: 6. In certain embodiments, a first fluorophore is conjugated to the thiol group of the cysteines (C) of SEQ ID NO: 6 and a second fluorophore is conjugated to the amino group of the lysines (K) of SEQ ID NO: 6. In some embodiments, the first fluorophore (conjugated to cysteine) is the donor fluorophore and the second fluorophore (conjugated to lysine) is the acceptor fluorophore. In other embodiments, the first fluorophore (conjugated to cysteine) is the acceptor fluorophore and the second fluorophore (conjugated to lysine) is the donor fluorophore. In some of these embodiments, the donor fluorophore comprises Cy5.5 and the acceptor fluorophore comprises Cy7.

C. Polyanionic Carrier Macromolecules

Carrier polynucleotides (e.g., plasmid DNA, genomic DNA) have been added to polynucleotide complexes to aid in the condensation and overall stabilization of the complex and enhance the delivery efficiency of polynucleotides, such as siRNA (Li et al. (2008) Mol. Ther. 16:163-169; Li et al. (2008) J. Control. Rel. 126:77-84). Carrier polynucleotides are those polynucleotides that can be used in delivery systems to enhance the delivery of a bioactive compound (e.g., a polynucleotide of interest) to a cell. Generally, unlike a polynucleotide of interest, once delivered to a cell, the carrier polynucleotide does not exert a desired phenotypic change in a cell (e.g., a therapeutic effect, expression of a polypeptide of interest, a change in the expression level of an endogenous gene when delivered to a cell in vitro or in vivo). Non-limiting examples of carrier polynucleotides are genomic DNA (e.g., calf thymus DNA) and bacterially produced plasmid DNA. When administered to humans, carrier polynucleotides (e.g., bacterially-produced plasmid DNA, foreign genomic DNA, such as calf thymus DNA) may cause toxicity due to their immunogenicity (Schwartz et al. (1997) J. Clin. Invest. 100:68-73). Plasmid DNA, for example, contains a large amount of unmethylated CpG motifs and therefore, formulations comprising plasmid DNA can be highly immunogenic (Li et al. (2008) Mol. Ther. 16:163-169; Li et al. (2008) J. Control. Rel. 126:77-84). Thus, the presently disclosed subject matter provides non-polynucleotide polyanionic carrier macromolecules that are useful for the delivery of bioactive compounds with delivery systems. The delivery systems presented herein can comprise polyanionic carrier macromolecules that are encapsulated, along with a bioactive compound, within a lipid vehicle (e.g., cationic liposome). In some embodiments, the polyanionic carrier macromolecule is not a carrier polynucleotide and the delivery systems are essentially free of carrier polynucleotides. As used herein when referring to carrier polynucleotides, the term “essentially free” is intended the delivery system does not comprise a sufficient amount of carrier polynucleotides to stimulate a significant or detectable immune response when the delivery system is administered to a subject.

In general, polyanionic carrier macromolecules encapsulated within cationic liposomes of delivery systems facilitate condensation and stabilization of the delivery system complexes due to the charge-charge interaction between the multivalent polyanionic carrier macromolecule and the cationic liposome. As used herein, the term “polyanionic carrier macromolecule” refers to any molecule that carries more than one negative charge at physiological pH (e.g., multivalent) that can interact with the cationic lipids of the cationic liposome of a polynucleotide delivery system through charge-charge interactions in such a way as to allow the polynucleotide delivery system to deliver the encapsulated bioactive compound to a cell, either in an in vitro or in vivo system. The ability of a polynucleotide delivery system to deliver an encapsulated bioactive compound to a cell can be measured using assays described elsewhere herein (see, for example, Experimental Examples 2 and 3). In some embodiments, the polyanionic carrier macromolecule has a molecular weight of between about 5 and about 20,000 kDa, including but not limited to, about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, about 900 kDa, about 1,000 kDa, about 5,000 kDa, about 10,000 kDa, and about 20,000 kDa. In some embodiments, the polyanionic carrier macromolecule has a valence of between about 20 and about 100,000, including but not limited to, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 500, about 1,000, about 10,000, and about 100,000 at physiological pH. Non-limiting examples of polyanionic carrier macromolecules include polymers, such as polyanionic polysaccharides, polyanionic polypeptides, and combinations thereof (e.g., glycoproteins, proteoglycans), and polyanionic polynucleotides. The polyanionic carrier macromolecules may be naturally occurring (i.e., one existing in nature without human intervention), chemically synthesized, or in the case of polynucleotides, may be a recombinant polynucleotide (i.e., one existing only with human intervention).

In some embodiments, the polyanionic carrier macromolecule comprises a polyanionic carrier polysaccharide. The term “polysaccharide” refers to a carbohydrate molecule comprised of two or more monomers (monosaccharides) joined together by glycosidic bonds. Thus, disaccharides, trisaccharides, oligosaccharides, or any other term used to refer to a carbohydrate molecule comprising more than one monosaccharides linked together by glycosidic bonds are included within the definition of “polysaccharide,” and the term “polysaccharide” can be used instead of, or interchangeably with any of these terms. The term “polysaccharide” encompasses both homopolysaccharides (a polysaccharide comprising only one type of monosaccharide) and heteropolysaccharides (a polysaccharide comprising more than one type of monosaccharide). A heteropolysaccharide can include polysaccharides comprised of repeating disaccharide units, trisaccharide units, tetrasaccharide units, or a repeating unit of any length. A “polyanionic polysaccharide” is a polysaccharide comprising a multiplicity of negative charges at physiological pH. In some embodiments, the polyanionic polysaccharide comprises about 2 to about 200 monosaccharide units, including but not limited to, about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, and about 200 monosaccharide units. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% of the monosaccharide units that make up the polyanionic macromolecules have at least one negative charge. A polyanionic carrier polysaccharide is a polysaccharide that, once delivered to a cell, does not exert a desired phenotypic change in the cell.

In some embodiments, the polyanionic carrier polysaccharide comprises a polyanionic glycosaminoglycan. A glycosaminoglycan is an unbranched polysaccharide comprising repeating disaccharide units, wherein one of the monosaccharide units of the disaccharide comprises an amino sugar, including, but not limited to, D-galactosamine or D-glucosamine. In some embodiments, the other monosaccharide unit of the repeating disaccharide comprises an uronic acid, including but not limited to D-glucuronic acid (GlcA) or L-iduronic acid (IdoA). In general, at least one of the monosaccharide monomers of the disaccharide unit has a negatively charged side group (e.g., carboxylate, sulfate). In some of these embodiments, the glycosaminoglycan comprises heparin, heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, and dextran sulfate. In certain embodiments, the glycosaminoglycan comprises hyaluronic acid or heparin sulfate. In particular embodiments, the molecular weight of the heparin sulfate that serves as a carrier macromolecule is about 4500 Da.

In other embodiments, the polyanionic carrier macromolecule comprises a polyanionic carrier polypeptide. A “polyanionic polypeptide” is a polypeptide, as defined elsewhere herein, comprising a multiplicity of negative charges at physiological pH. Unlike a polypeptide of interest, a polyanionic carrier polypeptide does not exert a desired phenotypic change in a cell upon delivery to the cell. The polyanionic carrier polypeptides may comprise glutamic acid residues, aspartic acid residues, or both. In some embodiments, the polyanionic carrier polypeptide has an amino acid composition in which glutamic acid residues, aspartic acid residues or both residues comprise at least 30% of the amino acid residues of the polypeptide, at least 40% of the amino acid residues of the polypeptide, at least 50% of the amino acid residues of the polypeptide, at least 60% of the amino acid residues of the polypeptide, at least 70% of the amino acid residues of the polypeptide, at least 80% of the amino acid residues of the polypeptide, at least 90% of the amino acid residues of the polypeptide, at least 95% of the amino acid residues of the polypeptide, at least 96% of the amino acid residues of the polypeptide, at least 97% of the amino acid residues of the polypeptide, at least 98% of the amino acid residues of the polypeptide, at least 99% of the amino acid residues of the polypeptide, or at least 100% of the amino acid residues of the polypeptide.

In some embodiments, the polyanionic carrier polypeptide is from about 2 amino acids to about 100,000 amino acids in length, including but not limited to about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 500, about 1,000, about 10,000, about 50,000, and about 100,000 amino acids in length.

The polyanionic carrier polypeptides to be used in formulating the presently disclosed complexes can be provided as naturally occurring proteins, as a chemically synthesized polypeptide, as a recombinant polypeptide expressed from a nucleic acid sequence which encodes the polypeptide, or as a salt of any of the above polypeptides where such salts include, but are not limited to, phosphate, chloride and sulfate salts.

In certain embodiments, the polyanionic carrier macromolecule comprises a polyanionic proteoglycan or a polyanionic glycopeptide. A proteoglycan comprises a glycosaminoglycan covalently bound to a polypeptide. Polyanionic proteoglycans may be comprised of a polyanionic glycosaminoglycan bound to a non-polyanionic (e.g., neutral, cationic) or a polyanionic polypeptide. A glycopeptide comprises a monosaccharide or polysaccharide covalently bound to a polypeptide. Polyanionic glycopeptides may be comprised of a polyanionic polysaccharide bound to a non-polyanionic polypeptide, a non-polyanionic polysaccharide bound to a polyanionic polypeptide, or a polyanionic polysaccharide bound to a polyanionic polypeptide.

In some embodiments, the molar ratio of the polyanionic carrier macromolecule to the bioactive compound (e.g., polynucleotide of interest) (polyanionic carrier macromolecule:bioactive compound) comprises about 1:1 to about 100:1, including but not limited to about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and about 100:1.

The polyanionic carrier macromolecules (e.g., polyanionic carrier polysaccharides, polyanionic carrier polypeptides) described herein can be used in place of carrier polynucleotides in delivery systems to deliver a bioactive compound (e.g., polynucleotide of interest) to a cell. In some embodiments, the delivery systems comprising a cationic liposome encapsulating a bioactive compound and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the delivery system is essentially free of carrier polynucleotides, have a reduced immunogenic effect compared with a control delivery system comprising a carrier polynucleotide when the delivery system and the control delivery system are administered to a subject.

By “immunogenic” or “immunogenic effect” when referring to a delivery system is intended that at least one component of the delivery system is capable of eliciting an immune response when administered to a subject. By “immune response” is intended an alteration in the reactivity of a subject's immune system and can comprise an innate immune response, including, but not limited to, inflammation (e.g., local or systemic) and complement activation, an adaptive immune response, such as a humoral (e.g., antibody production) or a cell-mediated immune response, or development of immunological tolerance. In some embodiments, the immunogenic effect comprises inflammation. An immunogenic effect, including but not limited to inflammation, can be measured by any method known in the art, including those methods described elsewhere herein (for example, see Experimental Example 3).

Polynucleotides of large size or with a high percentage of unmethylated CpG repeats have been demonstrated to be immunogenic. Thus, in some embodiments of the present invention, the polynucleotide delivery system comprises a polynucleotide of interest having a length of less than about 1 kb, less than about 900 bp, less than about 800 bp, less than about 700 bp, less than about 600 bp, less than about 500 bp, less than about 400 bp, less than about 300 bp, less than about 200 bp, less than about 100 bp, less than about 50 bp, less than about 40 bp, less than about 30 bp, less than about 29 bp, less than about 28 bp, less than about 27 bp, less than about 26 bp, less than about 25 bp, less than about 24 bp, less than about 23 bp, less than about 22 bp, less than about 21 bp, less than about 20 bp, less than about 19 bp, less than about 18 bp, less than about 17 bp, less than about 16 bp, less than about 15 bp, less than about 14 bp, less than about 13 bp, less than about 12 bp, less than about 11 bp, or less than about 10 bp.

D. Polycation

In some embodiments, the presently disclosed delivery systems can further comprise a polycation, wherein the lipid vehicle (e.g., cationic liposome) encapsulates the polycation. Polycations can physically associate with an anionic bioactive compound (e.g., polynucleotide of interest) and/or a polyanionic carrier macromolecule by the attraction of opposite charges. In those embodiments wherein the delivery system comprises a cationic liposome, a polynucleotide of interest, a polycation, and optionally, a polyanionic carrier macromolecule, and the polycation physically associates with the polynucleotide of interest and optionally, the polyanionic carrier macromolecule, the resulting complex is referred to as a “lipid/polycation/polynucleotide nanoparticle,” “LPP,” “LPD,” “LPP-NP,” or “LPD-NP”. The polynucleotide component of the LPP nanoparticle can be DNA or RNA (see, for example, U.S. Pat. No. 5,795,587 and U.S. Pat. No. 6,008,202, herein incorporated by reference). In those embodiments in which the polyanionic carrier macromolecule of the LPP nanoparticle comprises hyaluronic acid or heparin sulfate, the complex is referred to as a “lipid/polycation/hyaluronic acid (or heparin sulfate) nanoparticle,” “LPH nanoparticle” or “LPH-NP.” In certain embodiments, the overall net charge of the LPP or LPH nanoparticle is positive at physiological pH.

Without being bound by any theory or mechanism of action, it is believed that the polycation physically associates with the negatively charged polynucleotide molecules, and serves to condense the polynucleotide, allowing the size of the liposome/polycation/polynucleotide complex to be reduced as compared to those polynucleotide delivery systems comprising polynucleotides that lack a polycation, and contributes to the stabilization of the overall complex. Likewise, polycations serve to associate with and condense other anionic bioactive compounds. In general, LPP and LPH nanoparticles are about 100 nm in diameter. In particular non-limiting embodiments, the LPP or LPH nanoparticles have a diameter of about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 125, about 150, about 175, about 200 nm, about 225 nm, or about 250 nm.

As used herein, a “polycation” refers to a macromolecule with many positively charged groups that are positively charged at physiological pH. The polycation can be selected from organic polycations having a molecular weight of between about 300 and about 200,000. In some embodiments, these polycations have a valence of between about 3 and about 1000 at pH 7.0. Polycations can include, but are not limited to, natural or synthetic amino acids, peptides, polypeptides, polyamines, carbohydrates and any synthetic cationic polymers. Non-limiting examples of polycations include polyarginine, polyornithine, protamines and polylysine, polybrene (hexadimethrine bromide), histone, cationic dendrimer, spermine, spermidine and synthetic polypeptides derived from SV40 large T antigen that have excess positive charges and represent a nuclear localization signal. In general, polycations that serve to condense the bioactive compound (e.g., polynucleotide) and stabilize the delivery system do not exert a desired phenotypic change in a cell upon delivery to the cell.

In some embodiments, the polycation is a polycationic polypeptide having an amino acid composition in which arginine residues comprise at least 30% of the amino acid residues of the polypeptide and lysine residues comprise less than 5% of the amino acid residues of the polypeptide. In certain embodiments, histidine, lysine and arginine together make up from about 45% to about 85% of the amino acid residues of the polypeptide and serine, threonine and glycine make up from about 10% to about 25% of the amino acid residues of the polypeptide. In still other embodiments, arginine residues constitute from about 65% to about 75% of the amino acid residues of the polypeptide and lysine residues constitute from about 0 to about 3% of the amino acid residues of the polypeptide.

In addition to the above recited percentages of arginine and lysine residues, the polycationic polypeptides can also contain from about 20% to about 30% hydrophobic residues, or about 25% hydrophobic residues. The polycationic polypeptide to be used in producing lipid/polycation/polynucleotide complexes can be up to about 500 amino acids in length, from about 20 to about 100 amino acids in length, or from about 25 to about 50 amino acids in length, and in other embodiments, from about 25 to about 35 amino acids in length.

In one embodiment, the arginine residues present in the polycationic polypeptide are found in clusters of 3-8 contiguous arginine residues or in clusters of 4-6 contiguous arginine residues.

In another embodiment, the polycationic polypeptide is about 25 to about 35 amino acids in length and about 65 to about 70% of its residues are arginine residues and 0 to 3% of its residues are lysine residues.

The polycationic polypeptides to be used in formulating the complexes of the invention can be provided as naturally occurring proteins, particularly certain protamines having a high arginine to lysine ratio as discussed above, as a chemically synthesized polypeptide, as a recombinant polypeptide expressed from a nucleic acid sequence which encodes the polypeptide, or as a salt of any of the above polypeptides where such salts include, but are not limited to, phosphate, chloride and sulfate salts. In certain embodiments, the polycation is protamine.

E. PEGylated Polynucleotide Delivery Systems

In some embodiments, the surface charge of the lipid vehicle (e.g., liposome) of the delivery system can be minimized by incorporating lipids comprising polyethylene glycol (PEG) moieties into the lipid vehicle. Reducing the surface charge of the lipid vehicle of the delivery system can reduce the amount of aggregation between the delivery system complexes and serum proteins and enhance the circulatory half-life of the complex (Yan, Scherphof, and Kamps (2005) J Liposome Res 15:109-139). Thus, in some embodiments, the exterior surface of the lipid vehicle of the delivery system comprises a PEG molecule. Such a complex is referred to herein as a PEGylated delivery system. In those embodiments wherein the lipid vehicle comprises a liposome, the outer leaflet of the lipid bilayer of the liposome of the delivery system comprises a lipid-PEG conjugate.

A PEGylated delivery system can be generated through the post-insertion of a lipid-PEG conjugate into the lipid bilayer of the liposome through the incubation of the delivery system with micelles comprising lipid-PEG conjugates, as known in the art and described elsewhere herein (Ishida et al. (1999) FEBS Lett. 460:129-133; Perouzel et al. (2003) Bioconjug. Chem. 14:884-898; see Experimental section). By “lipid-polyethylene glycol conjugate” or “lipid-PEG conjugate” is intended a lipid molecule that is covalently bound to at least one polyethylene glycol molecule. In some embodiments, the lipid-PEG conjugate comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG). As described immediately below, these lipid-PEG conjugates can be further modified to include a targeting ligand, forming a lipid-PEG-targeting ligand conjugate (e.g., DSPE-PEG-AA). The term “lipid-PEG conjugate” also refers to these lipid-PEG-targeting ligand conjugates and a delivery system comprising a lipid vehicle comprising a lipid-PEG targeting ligand conjugate are considered to be both a PEGylated delivery system and a targeted delivery system, as described immediately below.

PEGylation of lipid vehicles enhances the circulatory half-life of the lipid vehicles by reducing clearance of the complex by the reticuloendothelial (RES) system. While not being bound by any particular theory or mechanism of action, it is believed that a PEGylated delivery system can evade the RES system by sterically blocking the opsonization of the complexes (Owens and Peppas (2006) Int J Pharm 307:93-102). In order to provide enough steric hindrance to avoid opsonization, the exterior surface of the lipid vehicle (e.g., liposome) must be completely covered by PEG molecules in the “brush” configuration. At low surface coverage, the PEG chains will typically have a “mushroom” configuration, wherein the PEG molecules will be located closer to the surface of the liposome. In the “brush” configuration, the PEG molecules are extended further away from the liposome surface, enhancing the steric hindrance effect. However, over-crowdedness of PEG on the surface may decrease the mobility of the polymer chains and thus decrease the steric hindrance effect (Owens and Peppas (2006) Int J Pharm 307:93-102).

The conformation of PEG depends upon the surface density and the molecular mass of the PEG on the surface of the lipid vehicle. The controlling factor is the distance between the PEG chains in the lipid bilayer (D) relative to their Flory dimension, R_(F), which is defined as aN^(3/5), wherein a is the persistence length of the monomer, and N is the number of monomer units in the PEG (see Nicholas et al. (2000) Biochim Biophys Acta 1463:167-178, which is herein incorporated by reference). Three regimes can be defined: (1) when D>2 R_(F) (interdigitated mushrooms); (2) when D<2 R_(F) (mushrooms); and (3) when D<R_(F) (brushes) (Nicholas et al.).

In certain embodiments, the PEGylated delivery system comprises a stealth delivery system. By “stealth delivery system” is intended a delivery system comprising a lipid vehicle wherein the exterior surface of the lipid vehicle (e.g., the outer leaflet of the lipid bilayer of a liposome) comprises a sufficient number of lipid-PEG conjugates in a configuration that allows the delivery system to exhibit a reduced uptake by the RES system in the liver when administered to a subject as compared to non PEGylated delivery systems. RES uptake can be measured using assays known in the art, including, but not limited to the liver perfusion assay described elsewhere herein (see Experimental Example 4). In some of these embodiments, the stealth delivery system comprises a lipid vehicle wherein the exterior surface of the lipid vehicle (e.g., the outer leaflet of the lipid bilayer of a cationic liposome) comprises PEG molecules, wherein said D<R_(F).

In those embodiments in which the PEGylated delivery system comprising a liposome as the lipid vehicle is a stealth polynucleotide system, the outer leaflet of the lipid bilayer of the cationic liposome comprises a lipid-PEG conjugate at a concentration of about 4 mol % to about 15 mol % of the outer leaflet lipids, including, but not limited to, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, and about 15 mol % PEG. In certain embodiments, the outer leaflet of the lipid bilayer of the cationic liposome of the stealth delivery system comprises about 10.6 mol % PEG.

The polyethylene glycol moiety of the lipid-PEG conjugate can have a molecular weight ranging from about 100 to about 20,000 g/mol, including but not limited to about 100 g/mol, about 200 g/mol, about 300 g/mol, about 400 g/mol, about 500 g/mol, about 600 g/mol, about 700 g/mol, about 800 g/mol, about 900 g/mol, about 1000 g/mol, about 5000 g/mol, about 10,000 g/mol, about 15,000 g/mol, and about 20,000 g/mol. In some embodiments, the lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In certain embodiments, the lipid-PEG conjugate comprises DSPE-PEG₂₀₀₀.

F. Delivery Systems with a Covalent Bilayer

In some embodiments, the presently disclosed delivery systems comprise a lipid vehicle comprising a covalent bilayer. As used herein, the term “covalent bilayer” refers to a bilayer as described herein, wherein at least two amphipathic molecules within the inner leaflet are bound to one another, at least two amphipathic molecules within the outer leaflet are bound to one another, or at least two amphipathic molecules within the inner leaflet are bound to one another and at least two amphipathic molecules within the outer leaflet are bound to another, wherein the amphipathic molecules are bound to one another through at least one covalent bond. In one embodiment, the covalent bilayer does not comprise covalent bonds between an amphipathic molecule within the inner leaflet and an amphipathic molecule within the outer leaflet. In other embodiments, the outer leaflet of the covalent bilayer does not comprise covalent bonds between the amphipathic molecules within the outer leaflet.

In some embodiments, the percentage of amphipathic molecules within the inner leaflet that are covalently bound to at least one other amphipathic molecule of the inner leaflet is between about 1% and about 100%, including but not limited to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%. In certain embodiments, the percentage of amphipathic molecules within the outer leaflet that are covalently bound to at least one other amphipathic molecule of the outer leaflet is between about 1% and about 100%, including but not limited to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%.

As used herein, the term “covalent bond” or “covalent interaction” refers to a chemical bond, wherein a pair of electrons is shared between two atoms. Two molecules are said to be chemically bound to one another when the molecules have at least one chemical bond between atoms that make up the molecules. One covalent bond between two molecules is therefore comprised of the sharing of one pair of electrons between an atom in one molecule with an atom in another molecule. Covalent bonds are in contrast to “non-covalent bonds” or “non-covalent interactions” that do not involve the sharing of pairs of electrons, but rather involve more dispersed variations of electromagnetic interactions, and can include hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bonds.

In some embodiments, where the lipid vehicle comprises a covalent bilayer, the bilayer comprises biocleavable covalent bonds. In these embodiments, the covalent bonds between (and/or within) the amphipathic monomers comprising the leaflets of the covalent bilayer comprise biocleavable covalent bonds. Biocleavable covalent bonds are those that can be cleaved or broken upon entering a cell or an organism. The presence and subsequent cleavage of biocleavable covalent bonds facilitates the release of the encapsulated bioactive compound.

In some embodiments, the biocleavable covalent bonds are those that are more specifically cleaved after entering the cell (intracellular cleavage). In some embodiments, the biocleavable covalent bonds are cleavable in acidic conditions like those found in lysosomes. One non-limiting example is an acid-sensitive (or acid-labile) hydrazone linkage, such as an acylhydrazone bond, as described by Greenfield et al. (1990) Cancer Res. 50, 6600-6607, which is herein incorporated by reference in its entirety. Another non-limiting example is certain linkages or bonds subject to hydrolysis that include various aldehyde bonds with amino or sulfhydryl groups. Also included are amide bonds such as when N-hydroxysuccinimide ester (NHS ester) reacts with amines. Another non-limiting example is an acid-labile maleamate bond (see Rozema et al. (2007) Proc Natl Acad Sci USA 104:12982-12987, which is incorporated herein by reference in its entirety). In certain embodiments, the biocleavable covalent bonds are cleavable by enzymes more commonly found in endosomes or lysosomes, including but not limited to lysosomal acid hydrolases.

In some embodiments, the lipid vehicle comprises a covalent bilayer, wherein the inner leaflet of the covalent bilayer comprises an amphipathic polymer. An “amphipathic polymer” refers to a molecule comprised of a polymeric backbone, wherein the polymeric backbone comprises repeating monomeric units bound to one another by at least one covalent bond. “Monomeric units” refer to a group of identical or substantially identical chemical molecules that can be covalently bound to each other to form a larger molecular weight polymer. The monomeric units of the polymeric backbone can comprise a side chain. As used herein, the term “side chain” refers to a branched chain of atoms that is covalently bound to the monomeric unit of the polymeric backbone, but is distinct from the atoms comprising the polymeric backbone chain. The polymeric backbone can be comprised of monomeric subunits, wherein about 10% to about 100% of the monomeric subunits comprise at least one side chain, including but not limited to, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the monomeric subunits.

In some embodiments, the monomeric units of the polymeric backbone are of identical chemical composition and structure and comprise identical side chains. In other embodiments, the monomeric units of the polymeric backbone can be identical, but the associated side chains differ among the monomeric units within a polymeric backbone.

In those embodiments wherein the covalent bilayer comprises an inner leaflet comprising an amphipathic polymer, the monomeric units of the polymeric backbone can be hydrophobic or hydrophilic. In some embodiments, the monomeric units of the amphipathic polymer are hydrophobic and the side chains of the monomeric units are hydrophilic. In other embodiments, the monomeric units of the polymeric backbone are hydrophilic and the side chains of the monomeric units are hydrophobic.

The amphipathic polymers may be generated using techniques known in the art. The covalent bonds between the monomeric subunits or between the monomeric subunits of the polymeric backbone and the side chains can be any type of covalent bond. As noted elsewhere herein, in some embodiments, the covalent bond is a biocleavable covalent bond. Non-limiting examples of amphipathic polymers include the amphipol molecules (short amphipathic polymers comprised of a hydrophilic backbone grafted with alkyl chains) disclosed in Pocanschi et al. (2006) Biochemistry 45:13954-13961 and Tribet, Audebert, and Popot (1996) Proc Natl Acad Sci USA 93:15047-15050, each of which are herein incorporated by reference in their entireties, having the structure of Formula (II), which is illustrated in FIG. 1. In some embodiments, the amphipol molecule used in the presently disclosed compositions and methods comprises A 8-75, whereas in other embodiments, A34-35, A8-35, or A34-75 are used. Another non-limiting example of amphipathic polymers useful for generating a covalent bilayer is the cationic acrylate polymer Eudragit® E100 (copolymer consisting of a 1:2:1 ratio of methyl methacrylate, N—N-dimethylaminoethyl methacrylate, and butyl methacrylate) described in Alasino et al. (2005) Macromolecular Bioscience 5:207-213, which is herein incorporated by reference in its entirety.

In certain embodiments, the lipid vehicle comprises a covalent bilayer, wherein the inner leaflet of the covalent bilayer comprises an alternating amphipathic polymer. As used herein, an “alternating amphipathic polymer” refers to a polymeric backbone comprising monomeric units covalently bound to one another, wherein the side chains of the monomeric units alternate between hydrophilic and hydrophobic side chains. The hydrophilic and hydrophobic side chains are covalently bound to the monomeric units of the polymeric backbone. The monomeric units of the polymeric backbone of the alternating amphipathic polymer can be hydrophobic or hydrophilic.

The terms “alternate” or “alternating” when referring to monomers with side chains or a polymer that comprises monomers with side chains can refer to an arrangement wherein about every other monomer (e.g., about every second monomer) comprises a hydrophilic side chain and the remaining monomers comprise a hydrophobic side chain. Alternatively, an “alternating amphipathic polymer” can comprise a polymer, wherein about every third monomer, about every fourth monomer, about every fifth monomer, about every sixth monomer, about every seventh monomer, about every eighth monomer, about every ninth monomer, or about every tenth monomer comprises a hydrophilic side chain, with the remaining monomers comprising a hydrophobic side chain.

In certain embodiments, the alternating amphipathic polymer comprises a polymer wherein about every third monomer, about every fourth monomer, about every fifth monomer, about every sixth monomer, about every seventh monomer, about every eighth monomer, about every ninth monomer, or about every tenth monomer comprises a hydrophobic side chain, with the remaining monomers comprising a hydrophilic side chain.

Alternatively, in some embodiments, the alternating amphipathic polymer can comprise an irregularly spaced arrangement of hydrophobic and hydrophilic side chains. Irregularly spaced arrangements are those comprised of monomers comprising a hydrophobic or hydrophilic side chain, wherein the spacing of hydrophobic and hydrophilic side chains within the polymer does not follow a specific pattern. In these embodiments, the inner leaflet of the lipid vehicle can comprise monomers, wherein about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the monomers comprise hydrophilic side chains, with the remaining monomers comprising hydrophobic side chains. In these embodiments, the relative amount and spacing of hydrophilic side chains to hydrophobic side chains is sufficient to allow the formation of the lipid vehicle and delivery of the encapsulated bioactive compound.

In some embodiments, the hydrophilic side chains of the alternating amphipathic polymer are cationic (have a positive charge at physiological pH). In some of these embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the side chains of the alternating amphipathic polymer comprise cationic hydrophilic side chains.

Two covalently bound monomeric units (a dimer) of the polymeric backbone, wherein one of the monomers comprises a hydrophilic side chain and the second monomer comprises a hydrophobic side chain form an amphipathic molecule. Althernatively, a group of covalently bound monomers, wherein at least one monomer comprises a hydrophilic side chain and at least one monomer comprises a hydrophobic side chain form an amphipathic molecule. In accordance with the definition of a covalent bilayer provided elsewhere herein, the inner leaflet of the bilayer thus comprises amphipathic molecules bound to one another by covalent bonds.

Non-limiting examples of a polymeric backbone useful in this invention are polyethylene glycol, polyglutamate, and polysaccharides, or other types of polymers known in the art. Non-limiting examples of hydrophilic side chains include those that comprise a polar or charged group, such as carboxylic, sulfato, amino, sulfhydryl, nitro, and hydroxyl groups. Non-limiting examples of hydrophobic side chains include acyl chains and long chain saturated and unsaturated aliphatic hydrocarbon groups.

The monomeric subunits within the polymeric backbone will be of a sufficient number to allow the bioactive compound to be encapsulated by the lipid vehicle and to permit the encapsulated bioactive compound to be delivered to the desired physiological site, tissue, or cell.

In some embodiments, the amphipathic polymer comprises cross-linked amphipathic lipids. The terms “cross-linkable or “cross-linked” refers to chemical moieties that can be (“cross-linkable”) or have been (“cross-linked”) covalently bound to another such moiety of the same or different chemical composition. In these embodiments, the monomeric units of the polymeric backbone comprise the hydrophilic head groups of the amphipathic lipids and the side chains of the monomeric units comprise hydrophobic side chains, such as an acyl chain.

Suitable methods for generating such lipid vehicles and delivery systems comprising the same are presented elsewhere herein.

G. Delivery Systems with a Supported Bilayer

The presently disclosed delivery systems can comprise a supported bilayer. Upon self-assembling, LPP nanoparticles comprise two bilayers, an outer bilayer encircling an inner supported bilayer. In the presence of high concentrations of lipid-PEG conjugates, the outer bilayer of the LPP nanoparticles is stripped off in the form of small particles. In contrast, the inner bilayer remains intact in the presence of high concentrations of lipid-PEG conjugates due to an enhanced stability attributed to the charge-charge interaction of the negatively charged polynucleotide and polyanionic carrier macromolecule in the aqueous core and the cationic lipids of the lipid bilayer of the cationic liposome. The stabilization imparted by the charge-charge interaction allows the inner bilayer to incorporate a high percentage of PEGylated lipid molecules while remaining intact, leading to a complete surface shielding and reduction in RES uptake of the nanoparticle. Likewise, LPH nanoparticles can comprise two bilayers resulting in a supported bilayer.

Any delivery system comprising a supported bilayer can be PEGylated through a post-insertion step to neutralize the surface charge and reduce RES uptake, limiting non-specific binding and enhancing the pharmacokinetic properties of the delivery system. By “supported bilayer” when referring to a delivery system is intended a bilayer, wherein one or both of the leaflets (e.g., inner leaflet, outer leaflet) of the lipid bilayer has been sufficiently stabilized through an electrostatic force or covalent bonding to allow the bilayer to remain intact while incorporating a high enough percentage of lipid-PEG conjugates to completely shield the surface charge (e.g., the zeta potential of the liposome has a zeta potential of about 0) or to reduce the RES uptake of the PEGylated delivery system when compared to a delivery system lacking PEG. A lipid bilayer is said to “remain intact” when there is not a substantial increase in the permeability of the lipid bilayer, which can be measured using techniques known in the art (Nicholas et al. (2000) Biochim. Biophys. Acta 1463:167-178), or a substantial increase in the small particle population (e.g., less than 50 nm, not including lipid-PEG micelles), which can be measured using techniques known in the art and presented elsewhere herein (e.g., dynamic light scattering, see Experimental Example 4).

In some embodiments, the supported bilayer comprises a “core supported bilayer.” By “core supported bilayer” is intended a lipid bilayer that has been stabilized through an electrostatic or covalent interaction between members of the lipid bilayer and the contents of the inner core of the lipid vehicle. A non-limiting example of a delivery system comprising a lipid vehicle comprising a core supported bilayer is the LPP nanoparticle described herein, wherein the negatively charged components of the liposome core (e.g., polynucleotide of interest, polyanioinic carrier macromolecule) interact with the cationic lipids of the cationic liposome through charge-charge interactions.

In some embodiments, the supported bilayer comprises a leaflet supported bilayer. As used herein, a “leaflet supported bilayer” comprises a bilayer that has been stabilized through electrostatic or covalent interactions between members of the leaflets that comprise the bilayer (e.g., inner leaflet, outer leaflet). A covalent leaflet supported bilayer refers to a covalent bilayer as described herein that is sufficiently stabilized through covalent bonding between at least two amphipathic molecules within the inner leaflet, between at least two amphipathic molecules within the outer leaflet, or between at least two amphipathic molecules within the inner leaflet and between at least two amphipathic molecules within the outer leaflet to allow the bilayer to remain intact while incorporating a high enough percentage of lipid-PEG conjugates to completely shield the surface charge (e.g., the zeta potential of the lipid vehicle has a zeta potential of about 0) or to reduce the RES uptake of the PEGylated delivery system when compared to a delivery system lacking PEG. The leaflet supported bilayer, wherein the stability is imparted by interactions between the members of the bilayer, is distinct from the core supported bilayer, wherein the stability is imparted by interactions between members of the bilayer and the components of the vehicle core. However, a delivery system could comprise a supported bilayer, wherein the support is imparted from both the interactions between components of the bilayer (a leaflet supported bilayer) and from interactions between the core components and the bilayer (a core supported bilayer).

It should be understood that in some embodiments of the invention, a delivery system comprising a leaflet supported bilayer can be one in which the members of the inner leaflet of the bilayer interact with members of the outer leaflet of the bilayer through electrostatic or covalent interactions. It should also be understood that the presently disclosed delivery systems include those complexes that comprise a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, and wherein the lipid vehicle comprises a leaflet supported bilayer. In some of these embodiments, the leaflet supported bilayer comprises a bilayer that has been sufficiently stabilized through electrostatic interactions between at least two amphipathic molecules within the inner leaflet, between at least two amphipathic molecules within the outer leaflet, or between at least two amphipathic molecules within the inner leaflet and between at least two amphipathic molecules within the outer leaflet to allow the bilayer to remain intact while incorporating a high enough percentage of lipid-PEG conjugates to completely shield the surface charge (e.g., the zeta potential of the lipid vehicle has a zeta potential of about 0) or to reduce the RES uptake of the PEGylated delivery system when compared to a delivery system lacking PEG.

It is to be understood that the presently disclosed delivery systems comprising a lipid vehicle that encapsulates a bioactive compound (e.g., polynucleotide, polypeptide, small molecule, drug) and is capable of delivering the bioactive compound to a cell or tissue, can comprise a lipid vehicle that comprises a supported bilayer. In some embodiments, the lipid vehicle of the delivery system comprises a core supported bilayer. These delivery systems comprise interactions (e.g., electrostatic, covalent) between the bioactive compound or other components within the core and components of the lipid bilayer sufficient to stabilize the bilayer. In some embodiments, the delivery system comprises a polynucleotide delivery system as described herein, including but not limited to, a LPP or LPH nanoparticle.

In some embodiments in which the delivery system comprises a core supported bilayer, the lipid vehicle comprises a polycationic lipid. By “polycationic lipid” is intended a cationic lipid as described herein that comprises more than one positively charged group at physiological pH. A non-limiting example of a polycationic lipid is DSGLA, which comprises three positively charged groups, and was described elsewhere herein and in International Application No. ______, entitled “Methods and Compositions Comprising Novel Cationic Lipids,” which was filed concurrently herewith. In the case of LPP nanoparticles, the strong positive charge of the polycationic lipid contributes to an enhanced stabilization of the inner bilayer of the cationic liposome as well as the delivery system complex as a whole.

H. Targeted Polynucleotide Delivery Systems

In some embodiments, the delivery system comprises a lipid vehicle, wherein the exterior surface of the lipid vehicle comprises a targeting ligand, thereby forming a targeted delivery system. In those embodiments wherein the lipid vehicle comprises a liposome, the outer leaflet of the liposome can comprise a targeting ligand. By “targeting ligand” is intended a molecule that targets a physically associated molecule or complex to a targeted cell or tissue. As used herein, the term “physically associated” refers to either a covalent or non-covalent interaction between two molecules. A “conjugate” refers to the complex of molecules that are covalently bound to one another. For example, the complex of a lipid covalently bound to a targeting ligand can be referred to as a lipid-targeting ligand conjugate.

Alternatively, the targeting ligand can be non-covalently bound to a lipid. “Non-covalent bonds” or “non-covalent interactions” do not involve the sharing of pairs of electrons, but rather involve more dispersed variations of electromagnetic interactions, and can include hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bonds.

Targeting ligands can include, but are not limited to, small molecules, peptides, lipids, sugars, oligonucleotides, hormones, vitamins, antigens, antibodies or fragments thereof, specific membrane-receptor ligands, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds. Non-limiting examples of targeting ligands include asialoglycoprotein, insulin, low density lipoprotein (LDL), folate, benzamide derivatives, and monoclonal and polyclonal antibodies directed against cell surface molecules. In some embodiments, the small molecule comprises a benzamide derivative. In some of these embodiments, the benzamide derivative comprises anisamide.

The targeting ligand can be covalently bound to the lipids comprising the lipid vehicle of the delivery system, including a cationic lipid, or a co-lipid, forming a lipid-targeting ligand conjugate. As described above, a lipid-targeting ligand conjugate can be post-inserted into the lipid bilayer of a liposome using techniques known in the art and described elsewhere herein (Ishida et al. (1999) FEBS Lett. 460:129-133; Perouzel et al. (2003) Bioconjug. Chem. 14:884-898; see Experimental section). Some lipid-targeting ligand conjugates comprise an intervening molecule in between the lipid and the targeting ligand, which is covalently bound to both the lipid and the targeting ligand. In some of these embodiments, the intervening molecule is polyethylene glycol (PEG), thus forming a lipid-PEG-targeting ligand conjugate. An example of such a lipid-targeting conjugate is DSPE-PEG-AA, in which the lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxyl (DSPE) is bound to polyethylene glycol (PEG), which is bound to the targeting ligand anisamide (AA). Thus, in some embodiments, the cationic lipid vehicle of the delivery system comprises the lipid-targeting ligand conjugate DSPE-PEG-AA.

By “targeted cell” is intended the cell to which a targeting ligand recruits a physically associated molecule or complex. The targeting ligand can interact with one or more constituents of a target cell. The targeted cell can be any cell type or at any developmental stage, exhibiting various phenotypes, and can be in various pathological states (i.e., abnormal and normal states). For example, the targeting ligand can associate with normal, abnormal, and/or unique constituents on a microbe (i.e., a prokaryotic cell (bacteria), viruses, fungi, protozoa or parasites) or on a eukaryotic cell (e.g., epithelial cells, muscle cells, nerve cells, sensory cells, cancerous cells, secretory cells, malignant cells, erythroid and lymphoid cells, stem cells). Thus, the targeting ligand can associate with a constituent on a target cell which is a disease-associated antigen including, for example, tumor-associated antigens and autoimmune disease-associated antigens. Such disease-associated antigens include, for example, growth factor receptors, cell cycle regulators, angiogenic factors, and signaling factors.

In some embodiments, the targeting ligand interacts with a cell surface protein on the targeted cell. In some of these embodiments, the expression level of the cell surface protein that is capable of binding to the targeting ligand is higher in the targeted cell relative to other cells. For example, cancer cells overexpress certain cell surface molecules, such as the HER2 receptor (breast cancer) or the sigma receptor. In certain embodiments wherein the targeting ligand comprises a benzamide derivative, such as anisamide, the targeting ligand targets the associated molecule to sigma-receptor overexpressing cells, which can include, but are not limited to, cancer cells such as small- and non-small-cell lung carcinoma, renal carcinoma, colon carcinoma, sarcoma, breast cancer, melanoma, glioblastoma, neuroblastoma, and prostate cancer (Aydar, Palmer, and Djamgoz (2004) Cancer Res. 64:5029-5035).

Thus, in some embodiments, the targeted cell comprises a cancer cell. The terms “cancer” or “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, “cancer cells” or “tumor cells” refer to the cells that are characterized by this unregulated cell growth. The term “cancer” encompasses all types of cancers, including, but not limited to, all forms of carcinomas, melanomas, sarcomas, lymphomas and leukemias, including without limitation, bladder carcinoma, brain tumors, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, endometrial cancer, hepatocellular carcinoma, laryngeal cancer, lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and thyroid cancer. In some embodiments, the targeted cancer cell comprises a lung cancer cell. The term “lung cancer” refers to all types of lung cancers, including but not limited to, small cell lung cancer (SCLC), non-small-cell lung cancer (NSCLC, which includes large-cell lung cancer, squamous cell lung cancer, and adenocarcinoma of the lung), and mixed small-cell/large-cell lung cancer.

I. Pharmaceutical Compositions

The delivery systems described herein are useful in mammalian tissue culture systems, in animal studies, and for therapeutic purposes. The delivery systems comprising a bioactive compound having therapeutic activity when expressed or introduced into a cell can be used in therapeutic applications. The presently disclosed subject matter therefore provides pharmaceutical compositions comprising the delivery systems described herein.

The presently disclosed compositions can be formulated for delivery, i.e., administering to the subject, by any available route including, but not limited, to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes. In some embodiments, the route of delivery is intravenous, parenteral, transmucosal, nasal, bronchial, vaginal, and oral.

The presently disclosed pharmaceutical compositions also can include a delivery system with a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds also can be incorporated into the compositions.

As one of ordinary skill in the art would appreciate, a presently disclosed pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringability exists. In some embodiments, the pharmaceutical compositions are stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols, such as manitol or sorbitol, or sodium chloride are included in the formulation. Prolonged absorption of the injectable formulation can be brought about by including in the formulation an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., delivery system) in the required amount in an appropriate liquid carrier with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. In certain embodiments, solutions for injection are free of endotoxin. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In those embodiments in which sterile powders are used for the preparation of sterile injectable solutions, the solutions can be prepared by vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions also can be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically or cosmetically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel, or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. Compositions for oral delivery can advantageously incorporate agents to improve stability within the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the presently disclosed compositions can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders, and the like, also can be used.

Systemic administration of the presently disclosed compositions also can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds also can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical or cosmetic carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Guidance regarding dosing is provided elsewhere herein.

1. Articles of Manufacture

The present invention also includes an article of manufacture providing a delivery system described herein.

The article of manufacture can include a vial or other container that contains a composition suitable for the present method together with any carrier, either dried or in liquid form. The article of manufacture further includes instructions in the form of a label on the container and/or in the form of an insert included in a box in which the container is packaged, for carrying out the method of the invention. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow the subject or a worker in the field to administer the pharmaceutical composition. It is anticipated that a worker in the field encompasses any doctor, nurse, technician, spouse, or other caregiver that might administer the composition. The pharmaceutical composition can also be self-administered by the subject.

II. Methods

The present invention provides methods for delivering a bioactive compound to a cell and for treating a disease or unwanted condition in a subject with a delivery system. Further provided herein are methods for making the presently disclosed delivery systems as well as methods for detecting apoptosis in a cell.

A. Methods of Delivery

The presently disclosed delivery systems comprising a lipid vehicle that encapsulates a bioactive compound can be used to deliver the bioactive compound to cells by contacting a cell with the delivery systems. In some embodiments, the method comprises contacting a cell with a polynucleotide delivery system comprising a cationic liposome encapsulating a polynucleotide of interest and a polyanionic carrier macromolecule that is not a carrier polynucleotide (and in some embodiments, a polycation), wherein the polynucleotide delivery system is essentially free of carrier polynucleotides, in order to deliver the polynucleotide of interest to the cell. As described elsewhere herein, the term “deliver” when referring to a composition of the invention (e.g., a polynucleotide) refers to the process resulting in the placement of the composition within the intracellular space of the cell or the extracellular space surrounding the cell. The term “cell” encompasses cells that are in culture and cells within a subject. The delivery of a polynucleotide into an intracellular space is also referred to as “transfection.”

The delivery of a bioactive compound (e.g., polynucleotide) to a cell can comprise an in vitro approach, an ex vivo approach, in which the delivery of the bioactive compound into a cell occurs outside of a subject (the transfected cells can then be transplanted into the subject), and an in vivo approach, wherein the delivery occurs within the subject itself.

In some embodiments wherein a polypeptide of interest is delivered to a cell by a delivery system, the polypeptide of interest comprises the sequence set forth in SEQ ID NO: 3. As demonstrated elsewhere herein (Experimental Example 6), the EV peptide is cytotoxic. Without being bound by any theory or mechanism of action, the cytotoxicity of the EV polypeptide is believed to be due to the ability of the EV polypeptide to mimic the catalytic domain of the epidermal growth factor receptor (EGFR) and inhibit the phosphorylation of various substrates of EGFR, including STAT5b. Therefore, the presently disclosed subject matter provides a method of killing a cell, wherein the method comprises contacting the cell with a delivery system comprising a lipid vehicle encapsulating the EV polypeptide (having the amino acid sequence set forth in SEQ ID NO: 3). Methods for detecting cell death are well known in the art and include, but are not limited to, the following in vitro assays: the MTT assay, a colorimetric assay, which measures the activity of mitochondrial reductase enzymes with the tetrazolium salt 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and similar assays using other tetrazolium salts and formazan dyes, such as XTT, and the WST assay; the Trypan blue (TB) assay, Sulforhodamine B (SRB) assay, and the clonogenic assay. Further, methods that are known in the art for measuring levels of cellular necrosis and apoptosis can be used to determine if a cationic lipid or drug has cytotoxic activity. Such methods for the detection of apoptosis include, but are not limited to, the TUNEL Assay, measuring caspase activity, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) activation, mitochondrial cytochrome C release, apoptosis-inducing factor (AIF) translocation, and Annexin-V staining. In some embodiments, the percentage of cells that are killed or exhibit a reduced rate of reproduction, cell division, or cellular growth upon contacting the delivery system comprising the EV peptide are in the range of about 1% to about 100%, including, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and any other such value between about 1% and about 100%.

In some embodiments wherein a polynucleotide is delivered by a polynucleotide delivery system, the cationic liposome of the polynucleotide delivery system comprises a cationic lipid and a co-lipid. In certain embodiments, the co-lipid is cholesterol.

The presently disclosed subject matter also provides methods for delivering a bioactive compound to a cell, wherein said methods comprise contacting a cell with a delivery system comprising a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, wherein the lipid vehicle comprises a core supported bilayer. In some embodiments, the core supported bilayer comprises a polycationic lipid. In some of these embodiments, the core supported bilayer of the lipid vehicle of the delivery system comprises a lipid-PEG conjugate. In some of these embodiments, the delivery system comprises a stealth delivery system. In certain embodiments, the outer leaflet of the lipid bilayer of the lipid vehicle of the delivery system comprises a targeting ligand, thereby forming a targeted delivery system, wherein the targeting ligand targets the targeted delivery system to a targeted cell.

In other embodiments, a bioactive compound is delivered to a cell using a delivery system comprising a covalent bilayer, including a covalent leaflet supported bilayer. In some of these embodiments, the covalent leaflet supported bilayer of the lipid vehicle of the delivery system comprises a lipid-PEG conjugate. In some of these embodiments, the delivery system comprises a stealth delivery system. In certain embodiments, the outer leaflet of the lipid bilayer of the lipid vehicle of the delivery system comprises a targeting ligand, thereby forming a targeted delivery system, wherein the targeting ligand targets the targeted delivery system to a targeted cell.

B. Methods of Treatment or Prevention

The delivery systems described herein comprising a bioactive compound can be used for the treatment of a disease or unwanted condition in a subject, wherein the bioactive compound has therapeutic activity against the disease or unwanted condition when expressed or introduced into a cell. In those embodiments wherein the bioactive compound comprises a polynucleotide, when the polynucleotide of interest is administered to a subject in therapeutically effective amounts, the polynucleotide of interest or the polypeptide encoded thereby is capable of treating the disease or unwanted condition.

As used herein, the terms “treatment” or “prevention” refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a particular infection or disease or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure of an infection or disease and/or adverse effect attributable to the infection or the disease. Accordingly, the method “prevents” (i.e., delays or inhibits) and/or “reduces” (i.e., decreases, slows, or ameliorates) the detrimental effects of a disease or disorder in the subject receiving the compositions of the invention. The subject may be any animal, including a mammal, such as a human, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

The disease or unwanted condition to be treated can encompass any type of condition or disease that can be treated therapeutically. In some embodiments, the disease or unwanted condition that is to be treated is a cancer. As described elsewhere herein, the term “cancer” encompasses any type of unregulated cellular growth and includes all forms of cancer. In some embodiments, the cancer to be treated is a lung cancer. Methods to detect the inhibition of cancer growth or progression are known in the art and include, but are not limited to, measuring the size of the primary tumor to detect a reduction in its size, delayed appearance of secondary tumors, slowed development of secondary tumors, decreased occurrence of secondary tumors, and slowed or decreased severity of secondary effects of disease.

It will be understood by one of skill in the art that the delivery systems can be used alone or in conjunction with other therapeutic modalities, including, but not limited to, surgical therapy, radiotherapy, or treatment with any type of therapeutic agent, such as a drug. In those embodiments in which the subject is afflicted with cancer, the polynucleotide delivery systems can be delivered in combination with any chemotherapeutic agent well known in the art.

When administered to a subject in need thereof, the delivery systems can further comprise a targeting ligand, as discussed elsewhere herein. In these embodiments, the targeting ligand will target the physically associated complex to a targeted cell or tissue within the subject. In certain embodiments, the targeted cell or tissue comprises a diseased cell or tissue or a cell or tissue characterized by the unwanted condition. In some of these embodiments, the delivery system is a stealth delivery system wherein the surface charge is shielded through the association of PEG molecules and the lipid vehicle further comprises a targeting ligand to direct the delivery system to targeted cells.

Delivery of a therapeutically effective amount of a delivery system comprising a bioactive compound can be obtained via administration of a pharmaceutical composition comprising a therapeutically effective dose of this agent. By “therapeutically effective amount” or “dose” is meant the concentration of a delivery system or a bioactive compound that is sufficient to elicit the desired therapeutic effect.

As used herein, “effective amount” is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times.

The effective amount of the delivery system or bioactive compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount can include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound or complex, and, if desired, the adjuvant therapeutic agent being administered along with the polynucleotide delivery system. Methods to determine efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population).

Polynucleotide delivery systems comprising carrier polynucleotides have been demonstrated to be immunogenic and even immunotoxic (Li et al. (2008) Mol. Ther. 16:163-169; Li et al. (2008) J. Control. Rel. 126:77-84). Thus, in some methods of the invention, the polynucleotide delivery systems comprising a cationic liposome encapsulating a polynucleotide of interest and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the polyanionic carrier macromolecule is not a carrier polynucleotide, and wherein the polynucleotide delivery system is essentially free of carrier polynucleotides, have a reduced immunogenic effect compared with a control polynucleotide delivery system comprising a carrier polynucleotide when the polynucleotide delivery system and the control polynucleotide delivery system are administered to a subject.

As described elsewhere herein, by “immunogenic” when referring to a polynucleotide delivery system is intended that at least one component of the polynucleotide delivery system is capable of eliciting an immune response when administered to a subject, including inflammation.

Upon the administration of a composition comprising a polynucleotide delivery system or a control polynucleotide delivery system to a subject, an immunogenic response can be measured through any method known in the art, including, but not limited to, measuring the systemic inflammatory response of a subject through the measurement of serum levels of pro-inflammatory cytokines. Pro-inflammatory cytokines can include, but are not limited to, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), monocyte chemoattractant protein-1 (MCP-1).

By “control polynucleotide delivery system” is intended a polynucleotide delivery system that comprises all of the same components as the polynucleotide delivery system with which it is being compared, except where otherwise noted. For example, in some embodiments, a polynucleotide delivery system comprising a cationic liposome encapsulating a polynucleotide of interest and a polyanionic carrier macromolecule that is not a carrier polynucleotide, and wherein the polynucleotide delivery system is essentially free of carrier polynucleotides, is being compared with a control polynucleotide delivery system comprising a cationic liposome encapsulating a polynucleotide of interest and a polyanioinic carrier macromolecule, wherein the polyanioinic carrier macromolecule comprises a carrier polynucleotide as the polyanioinic carrier macromolecule. In some of these embodiments, the carrier polynucleotide is present at an equimolar concentration in the control polynucleotide delivery system as the polyanionic carrier macromolecule in the polynucleotide delivery system to which it is being compared. In comparison studies, equivalent doses (or doses that have been demonstrated to result in equivalent circulating levels of the delivery system complex in the systemic circulation) of the polynucleotide delivery system and the control polynucleotide delivery system are administered to a subject, and the immunogenic effect of each is then measured.

The dose ratio between toxic (e.g., immunotoxic) and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the presently disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

The pharmaceutical formulation can be administered at various intervals and over different periods of time as required, e.g., multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, disorder, or unwanted condition, previous treatments, the general health and/or age of the subject, and other diseases or unwanted conditions present. Generally, treatment of a subject can include a single treatment or, in many cases, can include a series of treatments. Further, treatment of a subject can include a single cosmetic application or, in some embodiments, can include a series of cosmetic applications.

It is understood that appropriate doses of a compound depend upon its potency and can optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject can depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

One of ordinary skill in the art upon review of the presently disclosed subject matter would appreciate that the presently disclosed compounds and pharmaceutical compositions thereof, can be administered directly to a cell, a cell culture, a cell culture medium, a tissue, a tissue culture, a tissue culture medium, and the like. When referring to the delivery systems of the invention, the term “administering,” and derivations thereof, comprises any method that allows for the compound to contact a cell. The presently disclosed compounds or pharmaceutical compositions thereof, can be administered to (or contacted with) a cell or a tissue in vitro or ex vivo. The presently disclosed compounds or pharmaceutical compositions thereof, also can be administered to (or contacted with) a cell or a tissue in vivo by administration to an individual subject, e.g., a patient, for example, by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration) or topical application, as described elsewhere herein.

C. Methods for Making a Delivery System

The presently disclosed subject matter provides methods for making a delivery system. Methods for making a polynucleotide delivery system comprising a cationic liposome, a polynucleotide of interest, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the polyanionic carrier macromolecule does not comprise a carrier polynucleotide, wherein the cationic liposome encapsulates the polynucleotide of interest and the polyanionic carrier macromolecule, and wherein the polynucleotide delivery system is essentially free of carrier polynucleotides, comprise the steps of:

-   -   a) providing a cationic liposome; and     -   b) mixing the cationic liposome with a solution comprising a         polynucleotide of interest and a polyanionic carrier         macromolecule that is not a carrier polynucleotide, thereby         forming the polynucleotide delivery system. In some of these         embodiments, the solution further comprises a polycation. In         certain embodiments, step b) produces a cationic liposome         comprising a core supported bilayer. In some embodiments, the         cationic liposome comprises a polycationic lipid.

Also provided herein are methods for making a polynucleotide delivery system comprising a cationic liposome, a polynucleotide of interest, a polyanionic carrier macromolecule, and a polycation, wherein the polyanionic carrier macromolecule does not comprise a carrier polynucleotide, and wherein the cationic liposome encapsulates the polynucleotide of interest, the polyanionic carrier macromolecule, and the polycation, wherein the method comprises the steps of:

-   -   a) providing a cationic liposome;     -   b) mixing the cationic liposome with a polycation to form a         liposome/polycation solution;     -   c) mixing a polynucleotide of interest with a polyanionic         carrier macromolecule to form a polynucleotide/polyanionic         carrier macromolecule solution; and     -   d) mixing the polynucleotide/polyanionic carrier macromolecule         solution with the liposome/polycation solution, thereby forming         the polynucleotide delivery system;     -   wherein steps a) and b) can be performed before or after step         c).

In certain embodiments, step d) produces a cationic liposome comprising a core supported bilayer. In some embodiments, the cationic liposome comprises a polycationic lipid.

Methods for preparing liposomes are known in the art. For example, a review of methodologies of liposome preparation may be found in Liposome Technology (CFC Press NY 1984); Liposomes by Ostro (Marcel Dekker, 1987); Lichtenberg and Barenholz (1988) Methods Biochem Anal. 33:337-462 and U.S. Pat. No. 5,283,185. For example, cationic lipids and optionally co-lipids, from which the solvents have been removed, can be emulsified by the use of a homogenizer, lyophilized, and melted to obtain multilamellar liposomes. Alternatively, unilamellar liposomes can be produced by the reverse phase evaporation method (Szoka and Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198). In some embodiments, the liposomes are produced using thin film hydration (Bangham et al. (1965) J. Mol. Biol. 13:238-252). In certain embodiments, the liposome formulation can be briefly sonicated and incubated at 50° C. for a short period of time (e.g., about 10 minutes) prior to sizing (see Templeton et al. (1997) Nature Biotechnology 15:647-652).

The presently disclosed subject matter also provides methods for making a delivery system comprising a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, and wherein the lipid vehicle comprises a covalent bilayer, wherein the method comprises the following steps:

-   -   a) providing an amphipathic polymer in a solution comprising         water, an immiscible, volatile organic solvent, and a bioactive         compound, thereby forming a water-in-oil emulsion, wherein said         amphipathic polymer comprises a polymeric backbone, wherein said         polymeric backbone comprises a chain of repeating monomeric         units bound to one another by a covalent bond, wherein at least         10% of said monomeric units within said polymeric backbone         comprise a side chain, and wherein said amphipathic polymer is         selected from the group consisting of:         -   i) an amphipathic polymer, wherein said monomeric units of             said polymeric backbone comprise hydrophilic monomeric             units, and wherein said side chains of said monomeric units             comprise hydrophobic side chains;         -   ii) an amphipathic polymer, wherein said monomeric units of             said polymeric backbone comprise hydrophobic monomeric             units, and wherein said side chains of said monomeric units             comprise hydrophilic side chains; and         -   iii) an amphipathic polymer, wherein said monomeric units of             said polymeric backbone comprise hydrophobic or hydrophilic             monomeric units, and wherein said side chains alternate             between hydrophobic and hydrophilic side chains, thereby             forming an alternating amphipathic polymer;     -   b) mixing amphipathic lipids with said water-in-oil emulsion;         and     -   c) evaporating the volatile organic solvent, thereby forming the         delivery system.

In some embodiments, the lipid vehicle comprises a covalent bilayer, wherein the inner leaflet of the covalent bilayer comprises a plurality of amphipathic lipids, wherein the amphipathic lipids comprise cross-linked hydrophilic head groups, and wherein said hydrophilic head groups are bound to one another by a covalent bond.

“A plurality of amphipathic lipids” comprising a covalent bilayer may refer to a group of amphipathic lipids of the same chemical composition, a group of amphipathic lipids with the same hydrophilic head groups, but different hydrophobic portions (e.g., acyl chains), a group of amphipathic lipids with different hydrophilic head groups, but the same hydrophobic portions, or to those amphipathic lipids with different chemical compositions within the hydrophilic head groups and hydrophobic portions.

In some embodiments, the cross-linked amphipathic lipids of the inner leaflet are cationic, wherein the hydrophilic head groups and the amphipathic lipid overall is positively charged at physiological pH. In some of these embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the hydrophilic head groups are hydrophilic.

Another method for making a delivery system comprising a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, and wherein the lipid vehicle comprises a covalent bilayer, comprises the steps of:

-   -   a) mixing a plurality of amphipathic lipids, wherein the         amphipathic lipids comprise a cross-linkable hydrophilic head         group, in a solution comprising water, an immiscible, volatile         organic solvent, and a bioactive compound, thereby forming a         water-in-oil emulsion;     -   b) adding an initiator, thereby cross-linking the cross-linkable         head groups of said amphipathic lipids;     -   c) mixing amphipathic lipids with the water-in-oil emulsion; and     -   d) evaporating the volatile organic solvent, thereby forming the         delivery system.

As used herein, an “initiator” refers to a chemical molecule, such as an enzyme that facilitates or catalyzes the cross-linking of the cross-linkable hydrophilic head groups within the amphipathic lipids.

An emulsion is a dispersion of one liquid in a second immiscible liquid. The term “immiscible” when referring to two liquids refers to the inability of these liquids to be mixed or blended into a homogeneous solution. Two immiscible liquids when added together will always form two separate phases. As used herein, an immiscible organic solvent is one that is immiscible with water. Emulsions are essentially swollen micelles, although not all micellar solutions can be swollen to form an emulsion. Micelles are colloidal aggregates of amphipathic molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the nonpolar portions of the lipid molecules at the interior of the micelle and the polar portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) has a range from about 50 to about 100. The term “micelles” also refers to inverse or reverse micelles, which are formed in a nonpolar solvent, wherein the nonpolar portions are at the exterior surface, exposed to the nonpolar solvent and the polar portion is oriented towards the interior of the micelle.

An oil-in-water (O/W) emulsion consists of droplets of an organic compound (e.g., oil) dispersed in water and a water-in-oil (W/O) emulsion is one in which the phases are reversed and is comprised of droplets of water dispersed in an organic compound (e.g., oil). Thermodynamically stable emulsions are those that comprise a surfactant (e.g, an amphipathic molecule) and are formed spontaneously. The term “emulsion” can refer to microemulsions or macroemulsions, depending on the size of the particles. Droplet diameters in microemulsions typically range from about 10 to about 100 nm. In contrast, the term macroemulsions refers to droplets having diameters greater than about 100 nm.

It will be evident to one of skill in the art that sufficient amounts of water, organic solvent, and amphipathic molecules are added to the solution in step a) of the above-recited methods to form the water-in-oil emulsion.

The water-in-oil emulsion useful in preparing delivery systems as described herein comprises water and an immiscible, volatile organic solvent. As used herein, the term “volatile” refers to a property of a solvent that can be readily evaporated at ambient temperature and pressure. Non-limiting examples of an immiscible, volatile organic solvent include chloroform, ether, acetyl acetate, n-hexane, and dichloromethane. In some embodiments, the evaporating step of the methods comprises the use of a rotary evaporator.

In certain embodiments, the methods result in the production of a delivery system comprising a lipid vehicle comprising a covalent leaflet supported bilayer.

In some embodiments, the methods for making the delivery system can further comprise a sizing step, wherein the sizing step comprises selecting a population of the delivery systems based on the size (e.g., diameter) of the particles. The delivery systems can be sized using techniques such as ultrasonication, high-speed homogenization, and pressure filtration (Hope et al. (1985) Biochimica et Biophysica Acta 812:55; U.S. Pat. Nos. 4,529,561 and 4,737,323). Sonicating a delivery system complex suspension either by bath or probe sonication produces a progressive size reduction down to small vesicles less than about 0.05 microns in size. Vesicles can be recirculated through a standard emulsion homogenizer to the desired size, typically between about 0.1 microns and about 0.5 microns. The size of the delivery system complexes can be determined by quasi-elastic light scattering (QELS) (Bloomfield (1981) Ann. Rev. Biophys. Bioeng. 10:421-450). The average diameter can be reduced by sonication of delivery system complexes. Intermittent sonication cycles can be alternated with QELS assessment to guide efficient complex synthesis. Alternatively, delivery system complexes can be extruded through a small-pore polycarbonate membrane or an asymmetric ceramic membrane to yield a well-defined size distribution. Typically, a suspension is cycled through the membrane one or more times until the desired size distribution is achieved. The complexes can be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.

In certain embodiments, the methods of making the delivery system can further comprise a purification step after the final step, wherein the delivery system complexes are purified from excess free bioactive agents and free lipids. Purification can be accomplished through any method known in the art, including, but not limited to, centrifugation through a sucrose density gradient or other media which is suitable to form a density gradient. It is understood, however, that other methods of purification such as chromatography, filtration, phase partition, precipitation or absorption can also be utilized. In one method, purification via centrifugation through a sucrose density gradient is utilized. The sucrose gradient can range from about 0% sucrose to about 60% sucrose or from about 5% sucrose to about 30% sucrose. The buffer in which the sucrose gradient is made can be any aqueous buffer suitable for storage of the fraction containing the complexes and in some embodiments, a buffer suitable for administration of the complex to cells and tissues.

In certain embodiments wherein the delivery system comprises a polynucleotide delivery system that has a net positive charge and/or the liposome of the delivery system has a positively charged surface at physiological pH, the amounts and ratios of the cationic liposome, the polycation, the polyanionic carrier macromolecule, and the polynucleotide that are mixed are such that the polynucleotide to lipid ratio allows for the delivery system complex to have a net positive charge at physiological pH (see, for example, U.S. Pat. No. 7,335,509, which is herein incorporated by reference).

In some embodiments wherein the lipid vehicle comprises a liposome, a targeted delivery system or a PEGylated delivery system is made, wherein the methods further comprise a post-insertion step following the preparation of the liposome or following the last step of the methods, wherein a lipid-targeting ligand conjugate or a PEGylated lipid is post-inserted into the liposome. Liposomes comprising a lipid-targeting ligand conjugate or a lipid-PEG conjugate can be prepared following techniques known in the art, including but not limited to those presented herein (see Experimental section; Ishida et al. (1999) FEBS Lett. 460:129-133; Perouzel et al. (2003) Bioconjug. Chem. 14:884-898). The post-insertion step can comprise mixing the liposomes with the lipid-targeting ligand conjugate or a lipid-PEG conjugate and incubating the particles at about 50° C. to about 60° C. for a brief period of time (e.g., about 5 minutes, about 10 minutes). In some embodiments, the delivery systems are incubated with a lipid-PEG conjugate at a concentration of about 5 to about 20 mol %, including but not limited to about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol %, and about 20 mol %, to form a stealth delivery system. In some of these embodiments, the concentration of the lipid-PEG conjugate is about 10 mol %. The polyethylene glycol moiety of the lipid-PEG conjugate can have a molecular weight ranging from about 100 to about 20,000 g/mol, including but not limited to about 100 g/mol, about 200 g/mol, about 300 g/mol, about 400 g/mol, about 500 g/mol, about 600 g/mol, about 700 g/mol, about 800 g/mol, about 900 g/mol, about 1000 g/mol, about 5000 g/mol, about 10,000 g/mol, about 15,000 g/mol, and about 20,000 g/mol. In certain embodiments, the lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In some embodiments, the lipid-PEG conjugate comprises DSPE-PEG₂₀₀₀.

The present invention encompasses methods for making a delivery system comprising a lipid vehicle and a bioactive compound, wherein the lipid vehicle encapsulates the bioactive compound, wherein the lipid vehicle comprises a core supported bilayer, and where the core supported bilayer comprises a polycationic lipid, wherein the method comprises:

-   -   a) providing a lipid vehicle comprising a lipid bilayer, wherein         the lipid bilayer comprises a lipid bilayer, and wherein the         lipid bilayer comprises a polycationic lipid; and     -   b) mixing said lipid vehicle with a bioactive compound, thereby         forming said delivery system, wherein the lipid vehicle of the         delivery system comprises a core supported bilayer.

The method can further comprise a post-insertion step, as described above, wherein at least one of a lipid-targeting ligand conjugate and a lipid-PEG conjugate is post-inserted into the lipid bilayer of the lipid vehicle, wherein the post-insertion step is performed following step a) or after forming the delivery system.

C. Methods of Detecting Apoptosis

The presently disclosed subject matter provides for a method of detecting apoptosis in a cell through contacting the cell with a delivery system comprising a polypeptide of interest comprising at least one caspase 3 recognition motifs and a donor and acceptor fluorophore at a sufficient distance to allow FRET to occur between the two fluorophores and a means for delivering the polypeptide of interest into the cell, followed by the excitation of the donor fluorophore with an external light source and detection of the emission of the donor fluorophore and the acceptor fluorophore. An increase in the ratio of the emission of the donor fluorophore to the acceptor fluorophore in comparison to a control cell indicates apoptosis is occurring in the cell because the polypeptide of interest has been cleaved within the caspase 3 recognition motif through the activation of caspase 3 (which occurs during the initial stages of apoptosis), separating the two fluorophores and reducing the amount of FRET occurring between the two fluorophores. As used herein, a “control cell” is a comparable cell (e.g., similar tissue origin, similar phenotype, similar differentiation state, similar expression profile) that has not been contacted by the delivery system. In some embodiments, the increase in the ratio between the emission of the donor fluorophore to the acceptor fluorophore is between about 1% and about 1000%, including but not limited to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, and 1000%.

In some embodiments, a fluorometer, such as a nanofluorometer, is used to excite the fluorophore and detect the emitted light. In other embodiments, the cells are visualized using a fluorescent microscope and the pixel numbers of the fluorescing cells are quantitated to obtain the ratio of the emission of the donor fluorophore to the acceptor fluorophore.

The delivery of the imaging polypeptide can occur in vitro, ex vivo, or in vivo setting. Thus, a method of detecting apoptosis in a subject can comprise administering a delivery system to a subject, wherein the delivery system comprises a polypeptide of interest comprising at least one caspase 3 recognition motifs and a donor and acceptor fluorophore at a sufficient distance to allow FRET to occur between the two fluorophores and a means for delivering the polypeptide of interest into the cell, followed by the excitation of the donor fluorophore with an external light source and detection of the emission of the donor fluorophore and the acceptor fluorophore. The ratio of the donor and acceptor fluorophore can be measured using the IVIS® Kinetic live animal optical imaging system, which is available commercially from Caliper Life Sciences (Hopkinton, Mass.), or a similar system. In other embodiments, a multiphoton imaging system (e.g., two-photon imaging system, such as the system described in U.S. Pat. No. 7,282,514, which is herein incorporated by reference in its entirety), which has a higher sensitivity than the IVIS® system, can be used (for a review on multiphoton imaging systems, see, for example, Stutzmann and Parker (2005) Physiology 20:15-21, which is herein incorporated by reference in its entirety).

In some embodiments, the polypeptide of interest comprising at least one caspase 3 motif comprises the sequence set forth in SEQ ID NO: 6, wherein a first fluorophore is conjugated to the thiol group of the cysteines (C) and a second fluorophore is conjugated to the amino group of the lysines (K). In some embodiments, the first fluorophore (conjugated to cysteine) is the donor fluorophore and the second fluorophore (conjugated to lysine) is the acceptor fluorophore. In other embodiments, the first fluorophore (conjugated to cysteine) is the acceptor fluorophore and the second fluorophore (conjugated to lysine) is the donor fluorophore. In certain embodiments, the donor fluorophore comprises Cy5.5 and the acceptor fluorophore comprises Cy7. In some embodiments wherein the amino acid sequence of the polypeptide of interest comprises the sequence set forth in SEQ ID NO: 6, the amino terminus of the polypeptide is acetylated, which can contribute to the negative charge of the polypeptide.

In certain embodiments, the means for delivering the polypeptide of interest comprises a lipid vehicle that encapsulates the polypeptide of interest. In these embodiments, the polypeptide can have an overall negative charge (i.e., an anionic polypeptide). In some of these embodiments, the delivery system comprises a cationic liposome encapsulating the polypeptide of interest, a polyanionic carrier macromolecule (e.g., heparin sulfate), and a polycation (e.g., protamine sulfate). The surface charge of the liposome can be fully or partially shielded through the insertion of lipid-PEG conjugates into the outer leaflet of the lipid bilayer of the liposome. Further, the delivery system can be targeted to particular cells through the association of a targeting ligand with the liposome (e.g., through the post-insertion of lipid-targeting ligands or lipid-PEG-targeting ligand conjugates into the outer leaflet of the lipid bilayer of the liposome). Thus, the presently disclosed subject matter provides for the delivery of a polypeptide comprising at least one caspase 3 recognition motif and a donor and acceptor fluorophore at a sufficient distance to allow FRET to occur, wherein the polypeptide of interest is delivered to targeted cells. In some of these embodiments, the targeting ligand comprises anisamide and the targeted cells comprise a cancer (e.g., lung cancer). Therefore, the presently disclosed method provides for the real-time visualization of apoptosis occurring in a tumor cell, which can be utilized to tailor a treatment regimen or monitor the therapeutic efficacy of a given treatment regimen in a subject with cancer. In some embodiments, the polypeptide of interest has the amino acid sequence set forth in SEQ ID NO: 6.

In certain embodiments wherein the delivery system comprises a polypeptide of interest comprising at least one caspase 3 recognition motif and conjugated to a donor fluorophore and an acceptor fluorophore (i.e., an imaging polypeptide), the method can further comprise the administration or delivery of a cytotoxic bioactive compound. In some of these embodiments, the cytotoxic compound is delivered along with the imaging peptide in the same delivery system. Thus, the delivery system can function as a theranostic when delivered to a subject that would benefit from the cytotoxic properties of the bioactive compound (for example, a subject with cancer). As used herein, the term “theranostic” refers to the ability of a compound or complex to exert a therapeutic effect while also diagnosing the presence or progression of a particular disease or unwanted condition. For example, the theranostic delivery system comprising an imaging polypeptide comprising at least one caspase 3 recognition motif and a cytotoxic bioactive compound can be used to inhibit cell growth and induce apoptosis while simultaneously providing an image of those cells undergoing apoptosis in real time. In some of these embodiments, the cytotoxic bioactive compound comprises the EV peptide set forth in SEQ ID NO: 3. The theranostic delivery systems can be targeted to specific cells using a targeting ligand as described elsewhere herein, to allow for the specific simultaneous killing of a target cell and monitoring of the cell death.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL Materials and Methods for Examples 1-3 Materials

1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt (DSPE-PEG₂₀₀₀) were purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.). Protamine sulfate (fraction X from salmon), hyaluronic acid sodium salt from Streptococcus equi (HA) and calf thymus DNA (for hybridization, phenol-chloroform extracted and ethanol precipitated) were from Sigma-Aldrich (St. Louis, Mo.). Low molecular weight heparin (LMWH; Fraxiparin®, 4500 Da) was obtained from GlaxoSmithKline (Brentford, Middlesex, UK). DSPE-PEG₂₀₀₀-anisamide was synthesized in our lab using methods that were described previously (Banerjee et al. (2004) Int. J. Cancer 112:693-700).

Anti-luciferase siRNA (GL3) with the sense strand sequence of 5′-CUU ACG CUG AGU ACU UCG A-3′ (SEQ ID NO: 1) was purchased from Dharmacon (Lafayette, Colo.) in deprotected, desalted, annealed form. For the in vitro intracellular siRNA delivery study and determination of siRNA encapsulation efficiency, fluorescein-labeled siRNA (3′ end of the sense strand, FAM-siRNA) provided by Dharmacon was used.

B16F10 murine melanoma cells were obtained from the American Type Cell Collection and were stably transduced with the GL3 firefly luciferase gene using a retroviral vector. The gene silencing activity of various formulations comprising luciferase-targeted siRNA was assessed by analyzing luciferase activity. The cells were maintained in Dulbecco's modified eagle medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen). Sigma receptor-expressing B16F10 cells (Pham et al. (2007) J. Nucl. Med. 48:1348-1356) were used as model cells in our studies.

Experimental Animals

Female C57BL/6 mice (6-8 weeks old; 16-18 g) were purchased from Charles River Laboratories (Wilmington, Mass.). All work performed on animals was in accordance with and approved by the IACUC committee at UNC.

Optimization of the LPH-NP Formulation

Liposome-protamine-HA nanoparticles (LPH-NP) were prepared as follows. Briefly, small unilamellar liposomes consisting of DOTAP and cholesterol (molar ratio=1:1) were prepared by thin film hydration followed by membrane extrusion (ten times through a 400 nm membrane, ten times through a 200 nm membrane, ten times through a 100 nm membrane, and 15 times through a 50 nm membrane). The total lipid concentration of the liposomes was 40 mM. To prepare the siRNA/HA/protamine complex, 150 μl of protamine (200 μg/mL) and 150 μl of a mixture of siRNA and HA (160-210 μg/ml, weight ratio=1:1) were mixed in a 1.5 ml tube. The complex was allowed to stand at room temperature for 10 min before analysis of the size and zeta potential. The complex prepared by the optimal ratio of siRNA/HA and protamine was mixed with 0-50 μl of the prepared DOTAP/cholesterol liposomes (total lipid concentration=40 mM). Again, the particle size and zeta potential of the resulting particles were analyzed. Additionally, delivery efficiency of the LPH-NP of different lipid/siRNA ratios was determined as described herein below. The optimal ratio of the LPH-NP formulation was determined based on the particle size, zeta potential, and in vitro delivery efficiency. The optimized formulation was termed the naked LPH-NP. Non-targeted LPH-NP and targeted LPH-NP were prepared by incubating the naked LPH-NP suspension (330 μl) with 36.6 μl of a micellar solution of DSPE-PEG₂₀₀₀ or DSPE-PEG₂₀₀₀-anisamide (10 mg/ml) at 50° C. for 10 min, respectively, and then allowed to stand at room temperature for 10 min. The resulting LPH-NP formulations were used within 20 min for the following experiments.

Liposome-polycation-DNA nanoparticles (LPD-NP) were prepared as previously described (Li et al. (2008) J. Control. Rel. 126:77-84). Briefly, naked LPD-NP were obtained by quickly mixing 150 μl suspension A [8.3 mM liposomes (DOTAP/cholesterol molar ratio=1:1) and 200 μg/ml protamine] with 150 μl solution B (160 μg/ml siRNA and 160 μg/ml calf thymus DNA), followed by an incubation at room temperature for 10 min. Non-targeted LPD-NP and targeted LPD-NP were prepared by incubating the naked LPD-NP suspension (300 μl) with 37.8 μl of a micellar solution of DSPE-PEG₂₀₀₀ or DSPE-PEG₂₀₀₀-anisamide (10 mg/ml) at 50° C. for 10 min, respectively, followed by a 10 min incubation at room temperature. The resulting LPD-NP formulations were used within 20 min for the following experiments.

The distribution of particle sizes of the samples was measured using a submicron particle sizer (NICOMP particle sizing systems, AutodilutePAT Model 370, Santa Barbra, Calif.) in the NICOMP mode. The polydispersity index was also checked to evaluate distribution characteristics. The zeta potential of the samples diluted in 1 mM KCl was determined by the Zeta Plus zeta potential analyzer (Brookhaven Instruments Corporation, Holtsville, N.Y.).

Transmission electron microscope (TEM) images of the resulting LPH-NP and LPD-NP were acquired using a Phillips CM12 (FEI, Hillsboro, Oreg.). Briefly, freshly prepared nanoparticle samples (5 μl) were dropped onto a 300-mesh carbon-coated copper grid (Ted Pella, Inc., Redding, Calif.) and incubated for a short period (5 min) at room temperature. Grids were then stained with 1% uranyl acetate (40 μl) and wicked dry. All images were acquired at an accelerating voltage of 100 kV. Gatan DigitalMicrograph software was used to analyze the images.

The siRNA encapsulation efficiency of the LPH-NP and LPD-NP was determined by passing the FAM-siRNA containing formulations through a Sepharose CL4B size exclusion column (Pharmacia Biotech, Uppsala, Sweden). Unencapsulated FAM-siRNA was separated from encapsulated FAM-siRNA and the incorporation efficiency was determined as previously described (Li et al. (2006) Ann. N.Y. Acad. Sci. 1082:1-8).

In Vitro Intracellular siRNA Delivery Study

B16F10 cells (0.5×10⁵ cells/0.25 ml/well) were seeded in 48-well plates (Corning Inc., Corning, N.Y.) 20 h prior to treatment. Cells were treated with different formulations containing 500 nM FAM-siRNA in culture medium at 37° C. for 4 h. Cells were washed three times with PBS followed by incubation with 200 μl lysis buffer (0.3% Triton X-100 in PBS) at 37° C. for 0.5 h. The fluorescence intensity of 100 μl cellular lysate was determined with a plate reader (λex: 485 nm, λem: 535 nm) (PLATE CHAMELEON Multilabel Detection Platform, Bioscan Inc., Washington, D.C.). For the free ligand competition studies, cells were co-incubated with 50 μM haloperidol and the different formulations.

In Vitro Luciferase Gene Silencing Study

B16F10 cells (1×10⁵ cells/0.5 ml/well) were seeded in 24-well plates (Corning Inc., Corning, N.Y.) 20 h prior to treatment. Cells were treated with various formulations containing 250 nM siRNA in culture medium at 37° C. for 24 h. Cells were washed three times with PBS followed by incubation with 100 μl lysis buffer (0.05% Triton X-100 and 2 mM EDTA in 0.1 M Tris-HCl) at 37° C. for 0.5 h. Ten μl cellular lysate was mixed with 100 μl substrate (Luciferase Assay System, Promega Co., Madison, Wis.) and luminescence was measured with a plate reader. The protein concentrations of the samples were determined with a protein assay kit (Micro BCA™ assay kit, Pierce). The luciferase activity was normalized with the protein content and expressed as percent luminescence intensity relative to the untreated control.

In Vivo Luciferase Gene Silencing Study

C57BL/6 mice were i.v. injected with 2×10⁵ B16F10 cells via the tail vein. Seventeen days later, mice were given i.v. injections of anti-luciferase siRNA at the dose of 0.15 mg/kg alone or complexed in various formulations. Control siRNA with the sense strand sequence: 5′-AAU UCU CCG AAC GUG UCA CGU-3′ (SEQ ID NO: 2; Zhang et al. (2005) Clin. Cancer Res. 11:6261-6269) formulated in targeted LPH-NP or targeted LPD-NP was also prepared to verify that the silencing effect was sequence dependent. For the dose response studies, tumor bearing mice were i.v. injected with siRNA in targeted LPH-NP at doses of 0.0375, 0.075, 0.15, 0.45 and 1.5 mg/kg. After 24 h, mice were euthanized and the lungs were collected. The tumor nodules in the lung were isolated and homogenized in 300 μl lysis buffer (0.05% Triton X-100 and 2 mM EDTA in 0.1 M Tris-HCl) followed by centrifugation at 10,000 rpm for 10 min. Ten μl of the supernatant was mixed with 100 μl luciferase substrate and the luciferase activity was measured with a plate reader. The protein concentrations of the samples were determined by using a protein assay kit, as described above. The luciferase activity was normalized with the protein content and expressed as percent luminescence intensity relative to the untreated control.

Cytokine Induction Assay

C57BL/6 mice were i.v. injected with anti-luciferase siRNA formulated in the targeted LPH-NP or the targeted LPD-NP at various doses. Targeted LPD-NP formulated with plasmid DNA (pNGVL-Luc prepared by Bayou Biolabs) (Zhang et al. (2006) Mol. Ther. 13:429-437) instead of calf thymus DNA was prepared and used as a positive control. Two hours after the injections, blood samples were collected from the tail artery and incubated at room temperature for 0.5 h to allow for coagulation. Serum was obtained by centrifuging the clotted blood at 16,000 rpm for 20-40 min. Cytokine levels were determined with ELISA kits that detect IL6 and IL12 (BD Biosciences, San Diego, Calif.).

Statistical Analysis

Data are presented as the mean±SD. The statistical significance was determined with the analysis of variance (ANOVA). P values of <0.05 were considered significant.

Example 1 Development of the LPH-NP Formulation

We prepared the complex of siRNA/hyaluronic acid (HA) and protamine in different ratios and measured their particle size and zeta potential. As shown in FIG. 2, particle size and zeta potential changed according to the ratio of siRNA/HA to protamine. Large aggregates were found at the approximate ratio of 0.9 (siRNA/HA:protamine, weight ratio). At this ratio, a neutral complex was formed (zeta potential ˜0 mV), which tended to aggregate. An increase in the amount of protamine in the complex resulted in an increase in the zeta potential with a dramatic change between ratios 0.865-0.9625 (from −35 mV to 20 mV). We chose 1.0 as the optimal ratio, as the complex stayed negatively charged and had a relatively small size (˜150 nm).

Next, we mixed the siRNA/HA/protamine complex with different amounts of cationic lipid to prepare the naked LPH-NP and analyzed the size and zeta potential of the resulting particle. As mentioned earlier, a slightly excessive amount of the cationic lipid is required to obtain fully coated LPH-NP. At the ratio of 142 (lipid:siRNA, molar ratio), large aggregates with a neutral charge were detected (FIG. 3). Increasing the lipid/siRNA ratio increased the zeta potential of the resulting particle. To further investigate what ratio results in the optimal LPH-NP formulation, we encapsulated FAM-siRNA in the LPH-NP formulations prepared with different lipid/siRNA ratios and determined their in vitro delivery efficiency. As shown in FIG. 4, the formulation with the ratio of 1067 had the highest cellular delivery efficiency. Further increases of the cationic lipid decreased the delivery efficiency. This may be due to competitive binding of the excess cationic liposomes with the cells. At the optimal ratio, the particle size was around 120 nm and the zeta potential was about 45 mV. TEM examination confirmed that the size of the optimized LPH-NP was around 100 nm (FIG. 5).

PEGylated LPH-NPs, with or without the targeting ligand anisamide were prepared to maintain stability under physiological conditions and to allow selective delivery into cells expressing the sigma receptor (Li et al. (2006) Mol. Pharm. 3:579-588). Characteristics of the PEGylated formulations are summarized in Table 1.

TABLE 1 Characteristics of PEGylated LPH-NPs and LPD-NPs Encapsulation Particle size^(a)) Zeta potential^(a)) Efficiency^(b)) Formulations (nm) (mV) (%) Non-targeted LPH-NP 114.4 ± 16.2 22.4 ± 2.01 92.2 ± 3.2 Targeted LPH-NP 117.9 ± 15.7 27.5 ± 1.96 92.3 ± 1.2 Non-targeted LPD-NP 117.5 ± 17.1 21.1 ± 1.56 92.6 ± 3.2 Targeted LPD-NP 114.3 ± 16.7 25.4 ± 1.82 91.8 ± 0.6 ^(a))Data are representative data from the repeated measure of 3 samples. ^(b))Each value represents the mean ± S.D. (n = 3). PEGylated LPH-NPs were prepared by incubating the naked LPH-NP suspension (330 μl) with 36.6 μl micellar solution of DSPE-PEG₂₀₀₀ or DSPE-PEG₂₀₀₀-anisamide (10 mg/ml) at 50° C. for 10 min. Particle size, zeta potential and siRNA encapsulation efficiency were measured. PEGylated LPD-NPs were prepared as previously described (Li et al. (2008) Mol. Ther. 16:163-169; Li et al., Mol. Ther. in press). Notation of formulations: Non-targeted LPH-NP:PEGylated LPH-NP without anisamide; Targeted LPH-NP:PEGylated LPH-NP with anisamide; Non-targeted LPD-NP:PEGylated LPD-NP without anisamide; Targeted LPD-NP:PEGylated LPD-NP with anisamide.

The particle size was approximately 120 nm with a narrow size distribution (polydispersity index<0.2). PEGylation significantly reduced the zeta potential of the naked LPH-NP (a decrease from 45 mV to about 25 mV) because of the steric hindrance provided by the PEG. The zeta potential of the targeted LPH-NP was slightly higher than that of the non-targeted LPH-NP due to the positively charged anisamide ligand. The siRNA encapsulation efficiency of PEGylated LPH-NPs was greater than 90%. Overall, the characteristics of LPD-NP and LPH-NP were similar. Additionally, no differences between the LPH-NP and LPD-NP formulations were found in the TEM examination (data not shown).

Example 2 In Vitro Intracellular siRNA Delivery and Gene Silencing with Different Formulations

In vitro intracellular siRNA delivery studies were performed in B16F10 cells (FIG. 6). The fluorescence intensity of cells treated with free siRNA was indistinguishable from background fluorescence levels. This indicates that free siRNA inefficiently penetrates the cell membrane due to its highly hydrophilic nucleic acid backbone. The siRNA delivery efficiency of the targeted LPH-NP (PEGylated with ligand) was significantly higher than that of the non-targeted LPH-NP (PEGylated without ligand), and was inhibited by co-incubation with haloperidol, a known ligand for sigma receptor. This indicates that targeted LPH-NP can deliver siRNA to the B16F10 cells through sigma receptor mediated endocytosis, similar to the targeted LPD-NP (Li et al. (2008) J. Control. Rel. 126:77-84). Interestingly, the delivery efficiency of targeted LPH-NP was significantly higher than that of the targeted LPD-NP. The reason for this observation is not clear at the present time, but could be due to different rates of siRNA release from the LPD-NP and the LPH-NP.

In vitro gene silencing studies were performed in B16F10 cells, which were stably transduced with the firefly luciferase gene (FIG. 7). Free siRNA showed no gene silencing effect due to low membrane permeability (FIG. 7). The data were consistent with that of the in vitro intracellular siRNA delivery study (FIG. 7). The gene silencing effect of the targeted LPH-NP was similar to that of the targeted LPD-NP even though the siRNA delivery efficiency of the targeted LPH-NP was higher than that of the targeted LPD-NP (FIG. 7). This discrepancy could be due to saturation of the gene silencing effect. The silencing activity of the targeted LPH-NP leveled off at siRNA concentrations greater than 250 nM (data not shown).

Example 3 In Vivo Luciferase Gene Silencing and Associated Immunotoxicity of Different Formulations

In vivo gene silencing studies were performed in the B16F10 lung metastasis model (FIG. 6). Anti-luciferase siRNA formulated in targeted LPH-NPs silenced 80% of the luciferase activity (FIG. 8A). This effect was similar to that of anti-luciferase siRNA formulated in targeted LPD-NPs (FIG. 8A). The other control treatments, including free siRNA, siRNA in non-targeted LPD-NPs or non-targeted LPH-NPs and control siRNA in targeted LPD-NPs or LPH-NPs, showed no RNAi effect. The data shown in FIGS. 5 and 7 suggest that the enhanced gene silencing activity of the targeted LPH-NP is mainly due to the significantly improved tumor uptake. The ED50 of targeted LPH-NP was 0.075 mg/kg (FIG. 8B), the same as the targeted LPD-NP formulation. The optimal dose for maximal gene silencing (80%) was 0.15 mg/kg, and further increases in dosage did not result in greater activity (FIG. 8B). Both the in vitro and in vivo gene silencing data showed that LPD-NP and LPH-NP formulations were equivalent for gene silencing activity.

Systemic immunotoxicity studies were performed in C57BL/6 mice (FIG. 9). The immunotoxicity of the formulations was evaluated by their induction of the proinflammatory cytokines (IL6 and IL12) in the serum. Targeted LPH-NPs did not induce significant production of IL6 and IL12 over a wide dose range (0.15-1.2 mg/kg). On the other hand, the targeted LPD-NP containing calf thymus DNA significantly increased IL6 and IL12 levels at doses higher than 0.45 mg/kg. The data suggest that the therapeutic window of the targeted LPH-NP was greatly improved as compared to the targeted LPD-NP. Thus, the targeted LPH-NP formulation shows a greater potential for clinical use as compared to the targeted LPD-NP.

Materials and Methods for Example 4 Materials

1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) (FIG. 11), 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane chloride salt (NBD-DOTAP), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE), cholesterol, DSPE-PEG₂₀₀₀, and DSPE-PEG₂₀₀₀-carboxyfluorescein (DSPE-PEG-CF) were purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.). Protamine sulfate (fraction X from salmon sperm), calf thymus DNA (for hybridization, phenol-chloroform extracted and ethanol precipitated), and Sepharose CL 2B were from Sigma-Aldrich (St. Louis, Mo.). N,N-di-stearoyl-N-methyl-N-2[N′—(N6-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DSGLA) was synthesized in our lab (see International Application No. ______, entitled “Methods and Compositions Comprising Novel Cationic Lipids,” which was filed concurrently herewith). The structure of DSGLA is shown in FIG. 11.

Anti-luciferase siRNA (GL3) with the sense strand sequence of 5′-CUU ACG CUG AGU ACU UCG A-3′ (SEQ ID NO: 1) was purchased from Dharmacon (Lafayette, Colo.) in a deprotected, desalted, annealed form. Fluorescein (FAM)-labeled siRNA (3′ end of the sense strand) was used to evaluate the incorporation efficiency for siRNA into the LPD. Cy3-labeled siRNA was used for the isolated liver perfusion study.

Experimental Animals

Female C57BL/6 mice of age 6-8 weeks (16-18 g) were purchased from Charles River Laboratories (Wilmington, Mass.). All work performed with animals was in accordance with and approved by the IACUC committee at the University of North Carolina at Chapel Hill (UNC).

Preparation of siRNA-Containing LPD Nanoparticles

LPD was prepared as previously described (Li et al. (2007) J Control Release 126:77-84). Briefly, unmodified LPD were obtained by quickly mixing suspension A [8.3 mM liposomes (DOTAP:cholesterol=1:1, molar ratio) and 0.2 mg/mL protamine in 150 μL nuclease free water] with solution B (0.16 mg/mL siRNA and 0.16 mg/mL calf thymus DNA in 150 μL nuclease free water), followed by incubation at room temperature for 10 min. PEGylated LPD was prepared by incubating the LPD suspension (300 μL) with 37.8 μL micelle solution of DSPE-PEG (10 mg/mL) at 50° C. for 10 min. PEGylated LPD was allowed to stand at room temperature for 10 min. The charge ratio of the formulation was about 1:5 (−:+). The particle size was measured using a submicron particle sizer (NICOMP particle sizing systems, Autodilute^(PAT) Model 370, Santa Barbara, Calif.) in the NICOMP mode. The zeta potential of various LPD formulations diluted in 1 mM KCl was determined by using a Zeta Plus zeta potential analyzer (Brookhaven Instruments Corporation, Holtsville, N.Y.). PEGylated LPD was freshly prepared and used within 20 min for the following experiments. For size exclusion chromatography, either 10 mol % NBD-DOTAP labeled liposomes, 10 mol % DSPE-PEG-CF labeled DSPE-PEG or FAM-siRNA was used for the preparation of the PEGylated LPD. For the liver perfusion study, cy3-siRNA was used.

Negative-Stain Electron Microscopy

TEM images were acquired using a Phillips CM12 (FEI, Hillsboro, Oreg.). Briefly, freshly prepared formulations (5 μL) samples were dropped onto 300 mesh carbon-coated copper grids (Ted Pella, Inc., Redding, Calif.) and incubated for 5 min at room temperature. Grids were then stained with 1% uranyl acetate (40 μL) and wicked dry. All images were acquired at an accelerating voltage of 100 kV. Gatan Digital Micrograph software was used to analyze the images.

Size Exclusion Chromatography

Ten μL of the samples was loaded onto a Sepharose CL 2B column (1×10 cm) that had been pre-equilibrated with phosphate buffered saline (PBS). Eluted fractions (200-300 μL) were collected, diluted 1:1 in ethanol, and analyzed for fluorescence intensity by a plate reader (λex: 485 nm, λem: 535 nm) (PLATE CHAMELEON Multilabel Detection Platform, Bioscan Inc., Washington, D.C.).

Isolated Liver Perfusion Study

C57BL/6 mice were sacrificed and the interior vena cava was incised to allow the blood to flush out when 3 mL warm PBS was infused into the mouse liver through the portal vein. Cy3-siRNA-containing LPD formulations (300 μL) were incubated with 50 μL mouse serum at 37° C. for 10 min, and then diluted with PBS (final volume=1 mL). The complex was infused into the isolated liver via the portal vein. Finally, the liver was perfused with 3 mL warm PBS, excised, fixed in 3.6% paraformaldehyde in PBS overnight, and frozen sectioned (5 μm in thickness). Sections were washed with PBS, permeabilized with 0.1% Triton X-100 in PBS, stained with Alexa Fluor® 488 Phalloidin (Invitrogen, Eugene, Oreg.), mounted with DAPI-containing medium (Vectashield®, Vector Laboratories Inc., Burlingame, Calif.) and imaged using a Leica SP2 confocal microscope.

Statistical Analysis

Data are presented as the mean±SD. The statistical significance was determined by using the analysis of variance (ANOVA, one way) or the two-sided student t-test. P values of <0.05 were considered to be significant.

Example 4 Development of Stealth Nanoparticles Stabilized with a Core Supported Bilayer

We have demonstrated that unmodified LPD nanoparticles are comprised of two lipid bilayer membranes (Tan et al. (2002) Methods Mol. Med. 69:73-81; see FIG. 10). According to calculations based on the cryo-TEM picture (FIG. 10A), the ratio of the surface area of the inner bilayer and outer bilayer was 1:1.8, indicating that approximately 36.4% of the total lipids were located in the inner bilayer and 63.6% of the lipids were in the outer bilayer. The formation mechanism of the LPD nanoparticles has been proposed (FIG. 10C; Tan et al. (2002) Methods Mol. Med. 69:73-81). In this proposed mechanism, the cationic peptide interacts with the nucleic acid and forms a negatively-charged complex, which then interacts with the cationic liposomes via a charge-charge interaction. Due to the strong charge-charge interaction, the liposomes collapse onto the peptide-nucleic acid complex core. Two separate lipid bilayer membranes form on the surface of the LPD nanoparticle as a result of bilayer fusion and re-organization. In this model, the inner bilayer is directly in contact with the nanoparticle core and is supported and stabilized by the charge-charge interaction of the cationic lipids and the negatively charged nanoparticle core. We hypothesized that a nanoparticle with a supported bilayer tolerates a higher level of DSPE-PEG, which is a surfactant, better than a regular bilayer. This unique feature of LPD nanoparticles may provide us an opportunity to modify the formulation with a high amount of DSPE-PEG to achieve an enhanced surface shielding and thus improve the pharmacokinetic properties of the nanoparticle formulation.

DSPE-PEG has been used widely in lipid based nanoparticle formulations, such as liposomes, to increase the blood circulation time (Yan et al. (2005) J Liposome Res 15:109-139). It is also known that incorporation of too much DSPE-PEG will disrupt the integrity of the lipid membrane due to its detergent-like properties (Dos Santos et al. (2007) Biochim Biophys Acta 1768:1367-1377). This causes an increase in membrane permeability and drug release. Since unmodified LPD nanoparticles comprise two lipid bilayer membranes, incubation with DSPE-PEG may strip off the membranes from the nanoparticles and form micelles of smaller particle size. Here, we examined the stability of nanoparticle formulations after the addition of different amounts of DSPE-PEG by measuring their particle size distribution.

By using dynamic light scattering, only one narrow size distribution (around 100 nm) was revealed for all four formulations, i.e., liposomes and LPD composed of either DOTAP or DSGLA, before the addition of DSPE-PEG (FIG. 12). However, after the addition of DSPE-PEG, a population of smaller particles appeared in a dose-dependent manner for both DOTAP and DSGLA liposomes. It is noted that pure DSPE-PEG micelle was undetectable at the concentrations used in this experiment. Thus, the smaller size particles must have come from the nanoparticles upon the introduction of DSPE-PEG.

The light scattering method can quickly determine the stability of the nanoparticles. The LPD formulations showed significantly higher stability compared to the liposomes. When 10 mol % of DSPE-PEG was added, the LPD formulations remained relatively stable, while the liposome formulations showed a significant increase in the smaller size population (˜5%). The light scattering data (FIG. 12) is consistent with the TEM observation (FIG. 13), in which no distinct particles were detected in the DOTAP liposomes after PEGylation; only tubular mixed micelles were present. On the other hand, nanoparticles around 100 nm in diameter were still detectable in the PEGylated LPD formulation containing DOTAP (FIG. 13). It is also noted that DSGLA (contains 3 positive charges) containing-LPD nanoparticles showed an improved stability compared to DOTAP (contains 1 positive charge) containing-LPD nanoparticles (FIG. 12), suggesting that LPD was stabilized by the charge-charge interaction.

Dynamic light scattering is a convenient method to assess the relative stability of the particles. For example, smaller size particles were found in the TEM photographs of the PEGylated LPD (FIG. 13, arrow heads), while light scattering data showed no presence of smaller particles (FIG. 12). It is known that particles of larger sizes show significantly greater light scattering compared to smaller size particles at the same concentration. Nevertheless, the dynamic light scattering data provided a quantitative comparison of the relative stability of different nanoparticle formulations.

As can be seen in FIG. 13, after 10 mol % PEGylation, DOTAP liposomes had transformed into tubular micellar structures, indicating the instability of the formulation. In the PEGylated LPD, however, “sprouts” were found in some of the particles (FIG. 13, arrows), suggesting that the lipids in the surface of the LPD were being stripped off and becoming smaller particles (FIG. 13, arrow heads).

To further characterize the LPD nanoparticles, we used 10 mol % NBD-DOTAP-labeled liposomes, 10 mol % DSPE-PEG-CF-labeled DSPE-PEG or FAM-siRNA to prepare the PEGylated LPD nanoparticles. A sepharose CL 2B column was used to separate particles of different sizes. In this study, a neutral liposome formulation (DOPC/Cholesterol/NBD-PE=49/49/2, molar ratio, mean particle size around 100 nm) was used to calibrate the column. Cationic liposomes and unmodified LPD nanoparticles containing an excess positive surface charge formed aggregates in the elution medium (PBS) and thus, could not be studied by chromatography. As shown in FIG. 14A, pure DSPE-PEG micelles could be clearly separated from the 100 nm-nanoparticles by the size exclusion column. At least two particle populations were observed in the NBD-DOTAP-labeled PEGylated LPD (FIG. 14B). The first major peak coincided with that of the 100 nm-nanoparticles, and the second peak lay between the peaks of micelles and 100 nm-nanoparticles. In contrast, the PEGylated liposomes (NBD-DOTAP labeled) showed a less significant first peak but a smear of particle size distribution, suggesting the lipid membrane was disrupted in this formulation. The observation is consistent with the dynamic light scattering (FIG. 12) and TEM data (FIG. 13). Moreover, the AUC of the first peak of the PEGylated LPD (NBD-DOTAP labeled) was about 37.2% (37.2±4.6%, n=3). The data suggest that approximately 37.2% of the total lipids were associated with the nanoparticles, while the rest of the lipids were stripped off by the DSPE-PEG micelles and formed smaller particles (<100 nm).

As mentioned earlier, approximately 36.4% (36.4±3.2%, n=5) of the total lipids were located in the inner lipid bilayer of the LPD, suggesting that only the inner lipid bilayer stayed with the nanoparticles. The inner cationic lipid bilayer was in direct contact with the negatively charged surface of the nucleic acid/protamine core and therefore, was more tolerant to PEGylation compared to the outer bilayer. However, a further increase of the input DSPE-PEG to 20 mol % caused damage to the supported bilayer. Only 24.2% of the total cationic lipids remained associated with the nanoparticles (data not shown), indicating 35% of the lipids in the supported bilayer were removed. Although the inner bilayer of the LPD was stabilized by charge-charge interaction, it could only tolerate a finite amount of DSPE-PEG.

FIG. 14C shows that around 20.6% (20.6±3.4%, n=3) of the DSPE-PEG (DSPE-PEG-CF labeled) was incorporated with the nanoparticles (first peak) after heating, while about 80% of the DSPE-PEG formed smaller particles with the cationic lipids stripped off from the nanoparticles. FIG. 14D indicates that the nanoparticles eluted with the first peak containing 90% of siRNA, 37.2% of the total lipids and 20.2% of the input DSPE-PEG was the major contributor to siRNA delivery for the PEGylated LPD formulation.

Ten μL of the PEGylated LPD (loading amount for column) contained 0.71 μg siRNA, 36.71 nmole lipids and 3.99 nmole DSPE-PEG. The nanoparticles eluted in the first peak contained 0.64 μg siRNA, 13.66 nmole lipids and 0.81 nmole DSPE-PEG. Assuming that DSPE-PEG was inserted only into the outer leaflet of the supported bilayer and the lipid content in the outer and inner leaflet of the bilayer were the same, we calculated that 10.6 mol % of the outer leaflet was modified with DSPE-PEG. This led to a complete charge shielding, in which the zeta potential of the purified PEGylated LPD was −5.6±4.5 mV (FIG. 15).

To demonstrate that the DSPE-PEG was incorporated onto the LPD, as described previously, we conjugated an anisamide ligand (AA) to the distal end of the PEG chain and compared the delivery efficiency of the LPD-PEG and LPD-PEG-AA into sigma receptor-expressing cells (Li et al. (2007) J Control Release 126:77-84; Li et al. (2006) Mol Pharm 3:579-588). LPD-PEG-AA nanoparticles showed an enhanced siRNA delivery into the receptor-positive cells (4-fold increase) but not the receptor-negative cells, and the improved delivery was partially inhibited by an excess amount of the free ligand (Li et al. (2007) J Control Release 126:77-84; Li et al. (2006) Mol Pharm 3:579-588). These data suggest that DSPE-PEG and DSPE-PEG-AA are incorporated into the supported bilayer of the LPD.

As shown in FIG. 15, the zeta potential of the LPD, PEGylated LPD and purified PEGylated LPD were 43.2, 20.04, and −5.6 mV, respectively. Approximately 90% siRNA was encapsulated in the purified PEGylated LPD, which was a neutral delivery vehicle. A neutral carrier is desirable for in vivo drug delivery because of its improved pharmacokinetics and reduced non-specific interactions with cells or serum protein.

To further investigate if the PEGylated LPD showed reduced reticuloendothelial system (RES) uptake in the liver, we performed a liver perfusion assay. As shown in FIG. 16, 10 mol % PEGylated LPD showed little sinusoidal uptake in the liver, while 0 or 5 mol % PEGylated LPD had significant RES uptake. The data suggest that sufficiently PEGylated LPD showing reduced RES uptake in the liver may exhibit improved pharmacokinetics for delivering siRNA when i.v. administered. Indeed, PEGylated LPD delivered 70-80% injected dose (ID)/g of tissue into the NCI-H460 xenograft tumor 4 h after i.v. injection, while the liver showed only moderate uptake (˜10% ID/g) (Li et al. (2008) Mol Ther 16:163-169).

Here, we propose a model to describe the formation of the PEGylated LPD nanoparticles (FIG. 17). After the addition of DSPE-PEG micelles at 50° C., the micelles acted like detergents and stripped off the outer lipid bilayer of the LPD. In the intermediate phase, “sprouts” (outer bilayer being stripped off from the LPD) were formed and eventually broken down into smaller PEGylated lipid particles. The inner lipid bilayer was stabilized by charge-charge interaction and therefore, remained intact. Around 20% of the input DSPE-PEG was inserted into the outer leaflet of the supported bilayer. Approximately 10.6 mol % of the leaflet was modified with DSPE-PEG, which completely shielded the surface charge of the LPD. At such a high degree of PEGylation, the PEG was shown to be present on the surface in the brush mode (Garbuzenko et al. (2005) Chem Phys Lipids 135:117-129), suggesting the surface was fully protected. On the other hand, the cationic liposomes without a supported bilayer were not stable upon the challenge of DSPE-PEG micelles. The bilayer was stripped off from the liposomes and became smaller particles.

Depending on the surface density and molecular weight of the PEG grafted to the lipid bilayer, three PEG conformations can be identified (Kenworthy et al. (1995) Biophys J 68:1921-1936). Factors controlling the PEG conformation include the distance between the PEG chains in the lipid bilayer (D) and the Flory dimension, R_(F), which is defined as aN^(3/5) (a is the persistence length of the monomer, N is the number of monomer units in the PEG) (Nicholas et al. (2000) Biochim Biophys Acta 1463:167-178). Three regimes can be defined: (1) when D>2 R_(F) (interdigitated mushrooms); (2) when D<2 R_(F) (mushrooms); and (3) when D<R_(F) (brushes) (Nicholas et al.). For a 100 nm-liposome grafted with DSPE-PEG₂₀₀₀, PEG chains have been found to be arranged in the mushroom mode in the presence of <4 mol % DSPE-PEG; in the transition mode with 4-8 mol % modification; and in the brush mode when >8 mol % PEGylation (Garbuzenko et al.). The brush configuration ensures that the entire surface of NP is covered (Owens and Peppas (2006) Int J Pharm 307:93-102). However, over-crowdedness of the PEG on the surface may decrease the mobility of the polymer chains and thus decrease the steric hindrance effect (Owens and Peppas). Therefore, we believe that surface modification of PEG₂₀₀₀ slightly greater than 8 mol % is desirable for a stealth NP. In our formulation, the NP was 10.6 mol % modified with PEG₂₀₀₀, which falls within the optimal conditions.

Example 5 Development of Delivery Systems with a Covalent Bilayer

In one approach, we utilize an amphipathic polymer to prepare the nanoparticles (FIG. 18). Amphipathic polymers contain a backbone polymer structure with an alternative arrangement of hydrophilic and hydrophobic side chains. The polymer is used to prepare a water in oil nano-emulsion with a water-soluble drug in the aqueous phase. Organic solvent that is immiscible with water and volatile is used for the oil phase, such as chloroform and dichloromethane. Then, the reverse-phase evaporation method is performed in the presence of lipids and water to remove the solvent and coat the W/O nano-emulsion with a layer of lipids (Kirby and Gregoriadis (1984) J Microencapsul 1:33-45); and thus, the nanoparticle with a covalent bilayer is obtained.

Another approach involves using a lipid-like surfactant with a polymerizable head group to prepare the nano-emulsion encapsulating a water soluble drug in the aqueous phase (FIG. 19). Then, the surfactants surrounding the aqueous phase are polymerized by the addition of an initiator. Finally, the reverse-phase evaporation method is used to coat another lipid leaflet on the particle and the nanoparticle with a supported bilayer is formed. It is noted that only the inner leaflet of the bilayer is cross-linked and the outer leaflet is of high fluidity for post-insertion of DSPE-PEG.

Example 6 Intracellular Delivery of Peptides

Most existing peptide and protein drugs are designed to target extracellular or cell surface receptors. This is because the drugs can gain ready access to their targets without crossing a membrane barrier(s). There are vast amounts of molecular targets that are located in the intracellular compartments, however. For example, most of the protein kinases and phosphatases are intracellular proteins. Currently, only small molecule drugs have been developed to inhibit these targets. Macromolecules, such as peptides, can not gain access to the intracellular compartment unless they are delivered with a vector or fused to a cell-penetrating peptide, such as the HIV Tat peptide, which delivers its cargo non-specifically. These studies provide for the delivery of peptides to intracellular targets in human tumor cells in a xenograft model. Both therapeutic and imaging peptides can be delivered separately or combined. In the latter case, the delivery system is referred to herein as theranostic nanoparticles. These studies open the door to a new class of drugs, i.e., targeted intracellular peptides. The impact to the pharmaceutical industry and human health is profound.

Studies were initiated by selecting a highly anionic peptide that can block an important intracellular signaling event. The phosphorylation and activation of the signal transducer and activator of transcription 5b (STAT5b) by the epidermal growth factor receptor (EGFR) tyrosine kinase was chosen as the targeted signaling event. STAT proteins are cytoplasmic proteins that function as secondary messengers and transcription factors. The STAT proteins have a phosphotyrosine binding domain of src-homology 2 (SH2). The potential of the SH2 domain to interact with a number of signaling proteins allow the STATs to interfere with multiple cell signaling pathways (Mohamad and Mohammad (2006) JK-Practitioner 13(4):215-221). STAT activation has been implicated in human cancer cells, including the activation of upstream receptor tyrosine kinases, such as the EGFR, as well as nonreceptor kinases. STAT5b is related to very important genes for cell cycle, survival and proliferation such as c-myc, cyclin D, p21 and bcl-xL (Matsumura et al. (1999) EMBO J 18:1367-1377; Quelle et al. (1996) Mol Cell Biol 16:1622-1631; Matsumura et al. (1997) Mol Cell Biol 17:2933-2943). Activated STAT5b functions as a transcription factor for a number of cell cycle progression genes (Mui et al. (1996) EMBO J 15:2425-2433; Jr (1997) Science 277:1630-1635; Levy (2002) Nat Rev Mol Cell Biol 3:651-656; Chan et al. (2004) Cancer Res 64:2382-2389). If the peptide blocks the phosphorylation of Stat5b by EGFR, cells may undergo growth arrest or even apoptosis. The sequences of peptides that mimic the catalytic domain of EGFR were searched, resulting in the selection of a nonapeptide with the sequence of EEEE(pY)FELV (where pY stands for phosphotyrosine; SEQ ID NO: 3), which is referred to herein as the EV peptide for the amino acid residues at the amino and carboxyl termini of the peptide. EGFR tyrosine kinase is auto-phosphorylated at Y845 in the active site and the phosphorylated enzyme is highly active in phosphorylating several downstream signaling molecules including STAT5b (Sato, Aoto, and Fukami (1995) Biochem Biophys Res Commun 215(3):1078-1087; Biscardi et al. (1999) J Biol Chem 274:8335-8343; Boerner, Silva, and Parsons (2004) Mol Cell Biol 24:7059-7071). The chosen nonapeptide should bind STAT5b with high affinity and block its phosphorylation by EGFR, hence inducing cellular growth arrest and possibly apoptosis. Another nonapeptide with the scrambled sequence of E(pY)ELFEEVE (SEQ ID NO: 4), which is referred to herein as the EE peptide, was used as a control. In a cell-free system containing the lysate of H460 cells treated with EGF, the EV peptide, but not the control EE peptide, showed a dose-dependent inhibition of STAT5b phosphorylation with an apparent IC₅₀ of 2.5 μM (FIG. 20).

The peptide was then formulated in a nanoparticle formulation similar to the previously described LPD nanoparticle (Li and Huang (2006) Mol Pharm 3(5):579-588; Li (2008) Mol Ther 16(1):163-169; Li, Chono, and Huang (2008) Mol Ther 16(5):942-946; Li, Chono, and Huang (2008) J Control Release 126(1):77-84). Briefly, LPD nanoparticles are generated by mixing siRNA with a high molecular weight carrier polyanion such as calf thymus DNA (Li and Huang (2006) Mol Pharm 3(5):579-588; Li (2008) Mol Ther 16(1):163-169; Li, Chono, and Huang (2008) Mol Ther 16(5):942-946; Li, Chono, and Huang (2008) J Control Release 126(1):77-84), which is first complexed with a cationic polypeptide protamine sulfate to form negatively charged nanoparticle cores. Unilamellar cationic liposomes consisting of DOTAP/cholesterol (1:1, m/m) are then added to wrap the cores to form unmodified LPD, which can be incubated with a phospholipid-PEG conjugate containing the anisamide ligand at the distal end (referred to herein as LPD-PEG-AA nanoparticles). The final formulation contains about 10 mol % PEG lipid on the surface which effectively shields the cationic charges from DOTAP. The nanoparticle comprising the peptide has two modifications in comparison to the previously described LPD nanoparticles. Specifically, siRNA is replaced with the peptide and calf thymus DNA carrier was replaced with heparin. The resulting formulation is referred to herein as LPH-PEG for the anisamide targeted nanoparticles and LPH-PEG those lacking anisamide. The formulations were about 150 nm in diameter with an approximate zeta potential of 20 mV.

The uptake of the peptide by H460 cells was first studied in vitro. The EV peptide was fluorescently labeled with Alexa488 at the N-terminal α-NH₂ group, purified by gel filtration, and formulated in the nanoparticles. As can be seen in FIG. 21, the EV peptide formulated in LPH-PEG-AA was efficiently taken up by the H460 cells. There did exist some non-specific uptake of the EV peptide when it was formulated in the untargeted LPH-PEG nanoparticles, but the level of uptake was much less than those containing the AA targeting ligand. Free EV peptide was not taken up by H460 cells at all.

Next, the pharmacodynamics of the EV peptide was studied in vitro. H460 cells treated with LPH-PEG-AA containing EV peptide showed about 60% down-regulation of STAT5b phosphorylation as measured by an ELISA assay (FIG. 22). The EE control peptide did not show any significant effect, nor did the untargeted LPH-PEG formulation comprising the EV peptide. Therefore, under the conditions tested, the delivered EV peptide could partially block the phosphorylation of STAT5b in intact cells, suggesting that the peptide taken up by the tumor cells was at least partially bioavailable.

The effect of the delivered EV peptide on the growth of H460 cells was then investigated. Surprisingly, cells treated with LPH-PEG-AA containing the EV peptide showed complete growth arrest, whereas those treated with the same peptide formulated in the untargeted nanoparticles showed only a partial growth inhibition (FIG. 23). The free EV peptide had no effect. Cells treated with the EV peptide formulated in LPH-PEG-AA were much smaller in size than those treated with either the control EE peptide formulated in the same targeted nanoparticles or unformulated free peptide (FIG. 24, top row), suggesting apoptosis was the cause of the growth arrest. Also, cell nuclei in EV treated cultures displayed fragmented morphology, again indicating apoptosis (FIG. 24, bottom row). Indeed, greater than 70% of cells treated with the EV peptide formulated in LPH-PEG-AA underwent apoptosis as shown by flow cytometry using both annexin V and propidium iodide as markers (FIG. 25). Only a minor percentage (4-8%) of cells in the group treated with the EV peptide in the untargeted LPH-PEG formulation or those treated with the control EE peptide formulated in either the targeted or untargeted formulations were apoptotic. Again, free EV peptide was without any effect. These data clearly demonstrate the potent cytotoxic activity of the EV peptide when it is properly delivered.

To illustrate the potential of delivering this potent cytotoxic peptide to tumor cells in mice, the fluorescently labeled EV peptide formulated in either the targeted LPD-PEG-AA or the untargeted LPH-PEG nanoparticles were injected via the tail vein into nude mice bearing a subcutaneous H460 tumor. Four hours after the injection, mice were sacrificed and all major organs were taken for imaging using an IVIS camera. As can be seen in FIG. 26, the tumor from the mouse injected with the targeted formulation was bright in fluorescence. No significant uptake of the peptide was seen in other organs except a minor amount was found in the liver. Mice that received the same dose of the peptide formulated in the untargeted LPH-PEG nanoparticles also exhibited some tumor accumulation, but the level was significantly lower than the targeted formulation. The tumor was then homogenated and the fluorescent peptide was extracted for quantitation. Approximately 20% of the injected dose, or 33%/g tissue, was in the tumor. The tumor was about 0.6 g in weight. Assuming that about 50% of the tumor volume is occupied by tumor cells, and that the peptide was evenly distributed among all tumor cells, we estimate that the intracellular concentration of the delivered EV peptide is about 2 μM. This compares favorably with the IC₅₀ of the EV peptide, i.e., 2.5 μM, in the cell free assay of the p-STAT5b inhibition (FIG. 20). Thus, we believe by optimization of the injected dose and schedule, we should be able to reach the therapeutic level of accumulation of the EV peptide in the tumor.

To determine if the EV peptide that is delivered into the subcutaneous H460 tumor in the mice induces apoptosis, measurements of apoptosis using, for example a TUNEL assay or AIF immunostaining, can be performed. The TUNEL assay is based on the fact that apoptotic cells undergo DNA fragmentation (Negoescu et al. (1998) Apoptosi 52(6):252-258). This is a relatively late event in apoptosis. An earlier event is the translocation of apoptosis inducing factor (AIF). In healthy normal cells, AIF locates in the mitochondria (cytoplasm), but it translocates to the nucleus upon the initiation of apoptosis (Daugas et al. (2000) FASEB J 14:729-739). Both assays have been performed previously in the H460 tumor (Dao et al. (2003) Thorac Cardiovasc Surg 125:1132-1142).

The therapeutically effective doses of the EV peptide needed for therapeutic activity can be estimated using the subcutaneous H460 tumor mouse model. In previous studies with siRNA, the best therapeutic activity delivered by LPH-PEG-AA was three daily injections of 1.2 mg/kg siRNA per injection (Li et al. (2008) Mol Ther 16(1):163-169).

Example 7 Measurement of the Pharmacokinetics and Tissue Distribution of the EV Peptide

The pharmacokinetics (PK) and tissue distribution of the EV peptide are studied using a labeled peptide. In the experiments presented in Example 6, Alexa488-labeled peptide was used (FIG. 26). Quantitative measurement of fluorescence in the plasma or tissue requires the establishment of a standard curve for each tissue by using standard amounts of the labeled peptide added to the tissue or plasma homogenate, extracted, and measured by a fluorometer.

In addition to this method, ¹²⁵I-labeled peptide and heavy-amino acid labeled peptide are used as alternatives. EV peptide contains a phosphotyrosine (pY) group which is radioiodinated with very high specific radioactivity. Detection of the gamma radiation of the labeled peptide in the plasma or tissues requires no tissue preparation. Thus, PK and tissue distribution of the EV peptide are readily studied. However, measurement of radiolabeled peptide can not distinguish the intact peptide from the metabolized products. For this reason, the PK and tissue distribution work are also performed using heavy isotope labeled EV peptide. Peptides synthesized with stable isotope labeled amino acids are readily obtained through a commercial source. For example, ¹³C-labeled E, L and V can be used for a custom synthesis of the EV peptide. The labeled peptide and its possible metabolic products are readily quantitated by mass spectrometry. The extraction of the peptide from plasma and tissues follows protocols used in the art (see, for example, Houle et al. (1997) Analytical Biochemistry 250(2):162-168). The detection sensitivity of the labeled peptide is approximately in the pmole range, which is sufficient for the PK studies.

Judging from the clearance of the LPD-PEG-AA nanoparticles, which are very similar to the LPH-PEG-AA nanoparticles, the plasma clearance of the latter should follow a two phase standard clearance profile (Li et al. (2008) Mol Ther 16(1):163-169) with a t½ for the alpha phase of 0.2 h and the beta phase of 21 h. Thus, time points up to 8 h are used to establish the plasma clearance kinetics. The data is analyzed by a two compartment model using standard PK software (Winnolin®). Initially, three animals, per time point, per group are used to allow the detection of statistically significant differences between the groups (Li et al. (2008) Mol Ther 16(1):163-169).

Example 8 Development and Delivery of an Imaging Peptide that Monitors Apoptosis

In these studies, an imaging reporter system was developed which will allow the detection of apoptotic cells in a targeted tumor in situ upon administration of a cytotoxic agent. A peptide comprising an anionic tetrapeptide DEVD motif (SEQ ID NO: 5), which contains the sequence recognized by the activated caspase 3 (Nicholson et al. (1995) Nature 376(6535):37-43) was used for this purpose. The imaging peptide Ac-KDEVDCDEVDKDEVDC (SEQ ID NO: 6) contains three tetrapeptide repeats (underlined) to increase the amount of negative charges in the peptide for improved entrapment during the self-assembly of LPH nanoparticles. N-terminal acetylation further decreases the positive charge content of the peptide. Separating the caspase motifs are amino acids lysine (K) and cysteine (C), which were included to allow conjugation with two different fluorescent probes. Cy5.5 was conjugated to the thiol group of cysteine and Cy7 to the amino group of lysines. These two probes were chosen because they have excellent quantum yields and the emission spectrum of Cy5.5 (donor) overlaps significantly with the excitation spectrum of Cy7 (acceptor). The latter property is important for the fluorescence resonance energy transfer (FRET) that occurs when the two probes are in close proximity to each other. Thus, we expect that when the imaging peptide is intact, there will be significant FRET between the two probes and the ratio of Cy5.5/Cy7 emission should be low due to the energy transfer. When the peptide is cleaved, presumably by the activated caspase 3 as the result of apoptosis induction, the ratio should increase significantly due to the separation of the probes. FIG. 27 shows this was indeed the case. The fluorescence spectrum of the peptide was recorded with a nanofluorometer which only requires a 2 μL sample volume. As can be seen, the ratio of Cy5.5/Cy7 was 1.74 in the intact peptide (FIG. 27A). The ratio increased to 2.70 after the peptide was incubated with recombinant caspase 3 (FIG. 27B). This increase could be blocked by including a specific caspase 3 inhibitor DEVD-CHO in the incubation mixture (FIG. 27D), but not by the inclusion of BSA (FIG. 27C).

The double fluorescently-labeled imaging peptide was then formulated in the LPD-PEG-AA nanoparticles and incubated with H460 cells for 0.5 hours. After washing, the cells were further incubated with or without Taxol™ (50 μM) for 24 h to induce apoptosis before being examined by confocal microscopy. As can be seen in FIG. 28, incubated cells were fluorescent for both Cy5.5 and Cy7, indicating that the imaging peptide was taken up by the cells. The pixel numbers for individual cells (shown in red boxes) were quantitated to obtain the ratio of Cy5.5/Cy7 for each cell. The ratio was 0.663±0.045 for untreated cells, but increased significantly (p<0.001, n=10) to 0.813±0.104 for cells treated with Taxol™. It should be noted that the absolute value of the ratio depends on the confocal microscopy setting and could vary from experiment to experiment. This result indicates that the imaging peptide can be used to detect apoptosis in a quantitative manner.

Example 9 Monitoring Tumor Apoptosis with the Imaging Peptide In Vivo in a Nude Mouse H460 Xenograft Model

The imaging peptide is formulated in LPH-PEG-AA and other control nanoparticles and i.v. injected via the tail vein into nude mice bearing subcutaneous H460 tumor. Tumor apoptosis is induced by i.p. injection of Taxol™ at 20 mg/kg dose approximately 30 min after the injection of the nanoparticles. It is expected that the tumor accumulation of the imaging peptide will be at least 33% of the injected dose per gram of tumor tissue. An IVIS® Kinetic live animal optical imaging system is used to measure the Cy5.5/Cy7 ratio of the subcutaneous tumor. Alternatively, the two-photon imaging system, which has a higher sensitivity than the IVIS system, is used to measure the Cy5.5/Cy7 ratio. The animals will be anesthetized and the images of the tumor will be acquired as a function of time up to 3-4 h after the injection of Taxol™. Caspase activation is an early event of Taxol™ induced apoptosis. It is, therefore, expected that a significant increase in Cy5.5/Cy7 ratio will occur soon after the administration of Taxol™. Various doses of Taxol™ are used to generate a dose-response curve.

Example 10 Co-Formulating the Imaging Peptide with the Anti-Tumor EV Peptide in LPH-PEG-AA Nanoparticles

Since the imaging peptide is anionic, it can be co-formulated with the EV peptide described in Experimental Example 6, therapeutic siRNA, or other cytotoxic compounds, in the same nanoparticle formulation. Administration of these nanoparticle formulations should result in the induction of apoptosis in the tumor cells by the EV peptide, which can be reported by the imaging peptide delivered by the same nanoparticles. In other words, it will be a theranostic formulation that functions in real time.

The imaging peptide is co-formulated with the EV peptide to make the theranostic nanoparticles. The entrapment efficiency is determined using a Sepharose® CL-2B gel filtration column for both the imaging peptide and Alexa488-labeled EV peptide. The labeled EV and the imaging peptides are also used to demonstrate the co-delivery of both peptides to H460 cells using confocal microscopy. Since the delivery depends on the interaction of the AA ligand with the sigma receptor and the subsequent endocytosis, it is inhibitable by sigma antagonists such as haloperidol and inhibitors of endocytosis such as cytochalasin B. Co-delivery of both peptides is also the best method to study the kinetics of apoptosis induction by the EV peptide. At different time points after incubation, cells are fixed and measured for the fluorescence of Alexa488, Cy5.5 and Cy7. Cells high in the Alexa fluorescence are the ones that received a high dose of the EV peptide. These cells give an increase in Cy5.5/Cy7 ratio faster than the ones that received lower doses of the EV peptide. Thus, the experiment provides not only the kinetic data of EV-induced apoptosis, but also a dose-response curve.

To monitor tumor apoptosis in vivo in the nude mouse H460 xenograft model, the imaging peptide and EV peptide co-formulated theranostic LPH-PEG-AA nanoparticles are tail-vein injected into H460 nude mice xenograft model. The IVIS® Kinetic live animal imaging system is used to measure the Cy5.5/Cy7 ratio of the subcutaneous tumor. Alternatively, the two-photon imaging system, which has a higher sensitivity than the IVIS system, is used to measure the Cy5.5/Cy7 ratio. Again, since caspase-3 activation is an early event in apoptosis, the therapeutic effect of EV peptide could be shown both before and during the shrinkage of the solid tumor. To optimize the imaging activity of the “smart” nanoparticles, different amounts of the imaging peptide are co-formulated in the nanoparticles. Both the therapeutic and imaging effect is studied as a function of time and dose. To ascertain the interpretation of the imaging and therapeutic results, tissue sections of the tumor and other major organs are collected at different times after injection. The sections are stained for TUNEL and AIF to show apoptosis of the tumor cells. The presence of the imaging peptide in the tumor is observed by confocal microscopy.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. 

1. A delivery system comprising a cationic liposome, a bioactive compound, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein said cationic liposome encapsulates said bioactive compound and said polyanionic carrier macromolecule, wherein said cationic liposome comprises a lipid bilayer that comprises an inner leaflet and an outer leaflet, and wherein said delivery system is essentially free of carrier polynucleotides.
 2. (canceled)
 3. The delivery system of claim 1, wherein said polyanionic carrier macromolecule comprises a glycosaminoglycan selected from the group consisting of heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, and dextran sulfate. 4-22. (canceled)
 23. The delivery system of claim 1, wherein said outer leaflet comprises a lipid-PEG conjugate comprising a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol₂₀₀₀ (DSPE-PEG₂₀₀₀). 24-26. (canceled)
 27. The delivery system of claim 23, wherein said 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol₂₀₀₀ (DSPE-PEG₂₀₀₀) is conjugated to anisamide, thereby producing DSPE-PEG₂₀₀₀-AA. 28-30. (canceled)
 31. The delivery system of claim 1, wherein said bioactive compound comprises a polynucleotide of interest comprises a silencing element encoding an siRNA that targets a target polynucleotide selected from the group consisting of an oncogene and an epidermal growth factor receptor. 32-36. (canceled)
 37. The delivery system of claim 1, wherein said bioactive compound comprises a polypeptide of interest, and further wherein the polypeptide of interest comprises: (i) the amino acid sequence set forth in one of SEQ ID NO: 3 and SEQ ID NO: 6; (ii) at least one caspase 3 recognition motif; and (iii) a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest, wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source. 38-42. (canceled)
 43. The delivery system of claim 37, wherein said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or wherein said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue. 44-79. (canceled)
 80. A method of delivering a bioactive compound to a cell, said method comprising contacting a cell with the delivery system of claim
 1. 81. A method of treating a disease or unwanted condition in a subject, said method comprising administering a pharmaceutical composition comprising the delivery system of claim 1 and a pharmaceutically acceptable carrier to said subject, wherein said bioactive compound has therapeutic activity against said disease or unwanted condition.
 82. The method of claim 81, wherein said disease comprises a cancer. 83-100. (canceled)
 101. A method of detecting apoptosis in a cell, said method comprising: a) contacting said cell with a delivery system comprising a polypeptide of interest, wherein said polypeptide of interest has at least one caspase 3 recognition motif and a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest, wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source; b) exciting said donor fluorophore with a light source; and c) detecting the emission of said donor fluorophore and said acceptor fluorophore, wherein an increase in the ratio of the emission of said donor fluorophore to said acceptor fluorophore in comparison to a control cell indicates apoptosis in said cell. 102-103. (canceled)
 104. The method of claim 101, wherein said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue.
 105. (canceled)
 106. The method of claim 101, wherein said delivery system comprises a lipid vehicle that encapsulates said polypeptide of interest.
 107. The method of claim 106, wherein said delivery system further comprises a cytotoxic bioactive compound encapsulated by said lipid vehicle. 108-109. (canceled)
 110. The method of claim 106, further comprising a polyanionic carrier macromolecule, encapsulated by said lipid vehicle, and further wherein said polyanionic carrier macromolecule is selected from the group consisting of a polyanionic carrier polysaccharide, a polyanionic carrier polypeptide, a glycosaminoglycan, heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, dextran sulfate, and combinations thereof. 111-117. (canceled)
 118. The method of claim 106, wherein said lipid vehicle comprises an exterior surface comprising a polyethylene glycol (PEG) molecule.
 119. (canceled)
 120. The method of claim 118, wherein: (i) said PEG molecule comprises a lipid-PEG conjugate comprising a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glyco₂₀₀₀ (DSPE-PEG₂₀₀₀); (ii) said lipid vehicle comprises a liposome comprising an inner leaflet and an outer leaflet; and wherein (iii) said outer leaflet comprises the lipid-PEG conjugate at a concentration of about 8 mol % to about 12 mol % of the total lipids. 121-123. (canceled)
 124. The method of claim 106, wherein said lipid vehicle comprises an exterior surface comprising a targeting ligand that targets said delivery system to a targeted cell. 125-126. (canceled)
 127. The method of claim 124, wherein said 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol₂₀₀₀ (DSPE-PEG₂₀₀₀) is conjugated to anisamide, thereby producing DSPE-PEG₂₀₀₀-AA.
 128. The method of claim 124, wherein said targeted cell comprises a cancer cell selected from the group consisting of a bladder cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, an endometrial cancer, a hepatocellular carcinoma, a laryngeal cancer, a lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, and a thyroid cancer. 129-130. (canceled) 